Polynucleotides and polypeptides of the erythropoietin gene

ABSTRACT

The present invention relates to new polynucleotides deriving from the nucleotide sequence of the EPO gene and comprising new SNPs, new polypeptides derived from the natural EPO protein and comprising at least one mutation caused by the SNPs of the invention as well as their therapeutic uses.

RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 10/113,824 filed on Mar. 29, 2002 which claimsbenefit of U.S. Provisional Applications 60/358,598 filed on Feb. 21,2002, 60/345,440 filed on Jan. 4, 2002 and 60/343,163 filed on Dec. 21,2001. This application also claims foreign priority from Frenchapplication No. FR0104603 filed on Apr. 4, 2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to new polynucleotides deriving from thenucleotide sequence of the erythropoietin gene (EPO) and comprising newSNPs, new polypeptides derived from the natural erythropoietin proteinand comprising mutations caused by these SNPs as well as theirtherapeutic uses.

2. Related Art

The erythropoietin gene, hereinafter referred to as EPO, is described inthe publication Jacobs K. et al. (1985) “Isolation and characterizationof genomic and cDNA clones of human erythropoietin”; Nature 313 (6005),806-810.

The nucleotide sequence of this gene is accessible under accessionnumber X02158 in the GenBank database.

The erythropoietin is known to act on proliferation, differentiation,and maturation of progenitor cells of erythropoiesis. It determinestheir differentiation and maturation into erythrocytes.

EPO is also known to act as autocrine factor on cerain erythroleukemiccells and to be a mitogen and a chemoattractant for endothelial cells.

EPO is also known to stimulate activated and differentiated B-cells andto enhance B-cell immunoglobulin production and proliferation.

EPO synthesis is subject to a complex control circuit which links kidneyand bone marrow in a feedback loop. Synthesis depends on venous oxygenpartial pressure and is increased under hypoxic conditions.

EPO production is influenced also by a variety of other humoral factors,such as testosterone, thyroid hormone, growth hormone, andcatecholamines. In contrast, several cytokines such as IL-1, IL-6, andTNF-alpha, reduce EPO synthesis.

In the cell, binding of EPO to its receptor induces:

-   -   a release of membrane phospholipids,    -   the synthesis of diacyl glycerol,    -   an increase in intracellular calcium levels,    -   an increase in intracellular pH, and    -   an increase in intracellular phospholipase A2 and phospholipase        C, the latter inducing fos and myc oncogenes.

Excess of EPO is known to lead to erythrocytosis. This is accompanied byan increase in blood viscosity and cardiac output and may lead also toheart failure and pulmonary hypertension. A significant reduction ofplatelets is also observed.

Thrombosis is another adverse effect of an excess of EPO.

Pulmonary and cerebral embolism, i.e. the sudden obliteration of a bloodvessel by a clot or an extraneous compound transported by the blood,also constitutes a serious adverse effect related to EPO consumption.

However, when the amount of synthesized EPO is too low as it is in thecase of severe kidney insufficiencies, anemias are often observed. Thus,EPO is often administered to patients with severe kidney insufficiency,with hematocrit below 0.3, in particular in dialysis patients.

The most important complication in the treatment with EPO is hypertony,the increases in urea, potassium, and phosphate levels, an increase inblood viscosity, an expansion of thrombopoietic progenitor cells andcirculating platelets.

EPO is also used to activate erythropoiesis, allowing the collection ofautologous donor blood.

Moreover, EPO use has been suggested also for non-renal forms of anemiainduced, for example, by chronic infections, inflammatory processes,radiation therapy, and cytostatic drug treatment.

To a certain extent EPO is also a stimulating factor ofmegakaryocytopoiesis. The activity of EPO is synergized by IL-4.

EPO seems to possess neuroprotective capabilities since it has beendemonstrated that EPO protects neurons against cell death induced byischemia, probably by reducing free radicals production and by reducingoxidative stress effects.

It is known that the EPO gene is involved in different human disordersand/or diseases, such as different cancers like carcinomas, melanomas,myelomas, tumors, leukemia, and cancers of the liver, neck, head, andkidneys; cardiovascular diseases such as brain injury; metabolicdiseases such as those not related to the immune system like obesity;infectious diseases, in particular viral infections such as Hepatitis B,Hepatitis C, and AIDS; pneumonia; ulcerative colitis; central nervoussystem diseases such as Alzheimer's disease, schizophrenia, anddepression; tissue or organ graft rejection; wounds healing; anemia;allergy; asthma; multiple sclerosis; osteoporosis; psoriasis; rheumatoidarthritis; Crohn's disease; autoimmune diseases and disorders; genitalor venereal warts; gastrointestinal disorders; and disorders related totreatments by chemotherapy.

The inventors have found new polypeptide and new polynucleotide analogsto the EPO gene capable of having a different functionality from thenatural wild-type EPO protein.

These new polypeptides and polynucleotides can notably be used to treator prevent the disorders or diseases previously mentioned and avoid allor part of the disadvantages, which are tied to them.

BRIEF SUMMARY OF THE INVENTION

The invention has as its first object new polynucleotides that differfrom the nucleotide sequence of the reference wild-type EPO gene, inthat they comprise one or several SNPs (Single Nucleotide Polymorphism).

The nucleotide sequence SEQ ID NO. 1 of the human reference wild-typeEPO gene is composed of 3398 nucleotides and comprises a coding sequenceof 2149 nucleotides, from nucleotide 615 (start codon) to the nucleotide2763 (stop codon).

The EPO gene is composed of five exons whose positions on the nucleotidesequence SEQ ID NO. 1 are the following:

Exon 1: from nucleotide 397 to nucleotide 627 (comprises the start codonat position 615).

Exon 2: from nucleotide 1194 to nucleotide 1339.

Exon 3: from nucleotide 1596 to nucleotide 1682.

Exon 4: from nucleotide 2294 to nucleotide 2473.

Exon 5: from nucleotide 2608 to nucleotide 3327 (comprises the stopcodon at position 2763).

The applicant has identified 12 SNPs in the nucleotide sequence of thereference wild-type EPO gene.

These 12 SNPs are the following: 465-486 (deletion), c577t, g602c,c1288t, c1347t, t1607c, g1644a, g2228a, g2357a, c2502t, c2621g, g2634a.

It is understood, in the sense of the present invention, that thenumbering corresponding to the positioning of the SNP previously definedis relative to the numbering of the nucleotide sequence SEQ ID NO. 1.

The letters a, t, c, and g correspond respectively to the nitrogenousbases adenine, thymine, cytosine and guanine.

The first letter corresponds to the wild-type nucleotide, whereas thelast letter corresponds to the mutated nucleotide.

Thus, for example, the SNP g1644a corresponds to a mutation of thenucleotide g (guanine) at position 1644 of the nucleotide sequence SEQID NO. 1 of the reference wild-type EPO gene into a nucleotide a(adenine). The SNP 465-486 (deletion) corresponds to a mutation in whichthe 22 nucleotides from positions 465 to 486 of the nucleotide sequenceSEQ ID NO. 1 of the reference wild-type EPO gene have been deleted.

These SNPs have each been identified by the applicant using thedetermination process described in applicant's patent application FR 0022894, entitled “Process for the determination of one or severalfunctional polymorphism(s) in the nucleotide sequence of a preselectedfunctional candidate gene and its applications” and filed Dec. 6, 2000,cited here by way of reference.

The process described in this patent application permits theidentification of one (or several) preexisting SNP(s) in at least oneindividual from a random population of individuals.

In the scope of the present invention, a fragment of the nucleotidesequence of the EPO gene, comprising, for example, the coding sequence,was isolated from different individuals in a population of individualschosen in a random manner.

Sequencing of these fragments was then carried out on certain of thesesamples having a heteroduplex profile (that is a profile different fromthat of the reference wild-type EPO gene sequence) after analysis byDHPLC (“Denaturing-High Performance Liquid Chromatography”).

The fragment sequenced in this way was then compared to the nucleotidesequence of the fragment of the reference wild-type EPO gene and theSNPs in conformity with the invention identified.

Thus, the SNPs are natural and each of them is present in certainindividuals of the world population.

The reference wild-type EPO gene codes for an immature protein of 193amino acids, corresponding to the amino acid sequence SEQ ID NO. 2, thatwill be converted to a mature protein of 166 amino acids, by cleavage ofthe signal peptide that includes the first 27 amino acids.

The structure of the natural wild-type EPO protein comprises fourhelices called A, B, C, and D. The crystal structure of EPO complexedwith the EPO receptor indicates that only the three helices A, C, and Dare involved in EPO binding with its receptor (Syed et al. (1998).Efficiency of signaling through cytokine receptors depends critically onreceptor orientation. Nature 395:511-516). In addition, site directedmutagenesis studying the active site of EPO demonstrates that changes inamino acids situated in helix B have a limited effect on EPO activity(Eliott et al. (1997). Mapping of the active site of recombinant humanerythropoietin. Blood. 89: 493-502 ; Wen et al. (1994). Erythropoietinstructure-function relationships. Identification of functionallyimportant domains. J. Biol. Chem. 269:22839-22846).

Each of the coding SNPs of the invention, namely: g1644a, g2357a,c2621g, causes modifications, at the level of the amino acid sequence,of the protein encoded by the nucleotide sequence of the EPO gene.

These modifications in the amino acid sequence are the following:

The coding SNP g1644a causes a mutation of the amino acid aspartic acid(D) at position 70 in the immature protein of the EPO gene,corresponding to the amino acid sequence SEQ ID NO. 2, in asparagine (N)and at position 43 of the mature protein. In the description of thepresent invention, one will call the mutation encoded by this SNP D43Nor D70N according to whether one refers to the mature protein or to theimmature protein respectively.

The coding SNP g2357a causes a mutation of the amino acid glycine (G) atposition 104 in the immature protein of the EPO gene, corresponding tothe amino acid sequence SEQ ID NO. 2, in serine (S) and at position 77of the mature protein. In the description of the present invention, onewill call the mutation encoded by this SNP G77S or G104S according towhether one refers to the mature protein or to the immature proteinrespectively.

The coding SNP c2621g causes a mutation of the amino acid serine (S) atposition 147 in the immature protein of the EPO gene, corresponding tothe amino acid sequence SEQ ID NO. 2, in cysteine (C) and at position120 of the mature protein. In the description of the present invention,one will call the mutation encoded by this SNP S120C or S147C accordingto whether one refers to the mature protein or to the immature proteinrespectively.

The coding SNPs g1644a, g2357a, and c2621g, cause modifications of thespatial conformation of the polypeptides in conformity with theinvention compared to the polypeptide encoded by the nucleotide sequenceof the wild-type reference EPO gene.

These modifications can be observed by computational molecular modeling,according to methods that are well known to a person skilled in the art,making use of, for example, the modeling tools de novo (for example,SEQFOLD/MSI), homology (for example, MODELER/MSI), minimization of theforce field (for example, DISCOVER, DELPHI/MSI) and/or moleculardynamics (for example, CFF/MSI).

Examples of such models are given hereinafter in the experimentalsection.

1/ Computational molecular modeling indicates that the mutation D43N onthe mutated mature protein involves a structural modification of theloop located between helix A and helix B of the EPO protein, as well asa variation in the structure of the long loop connecting helices C and Dof the EPO protein in the area from P129 to I133 amino acids. Thoseresidues are located in front of the mutated amino acid N43. Since thismutation is located near the short helix F48-R53 involved in the bindingto the EPO receptor, it may have an effect on the interaction of the EPOprotein with its receptor. The D43 residue is highly conserved in allEPO orthologues. It could form salt bridges with positively chargedresidues (K45, R13 1), which are also conserved in EPO orthologues.

Thus, the mutated protein possesses a different three-dimensionalconformation from the natural wild-type EPO protein encoded by thewild-type EPO gene.

Computational molecular modeling also predicts that the presence of theasparagine amino acid at position 43 involves a significant modificationof the structure and of the function of the natural wild-type EPOprotein.

2/ Computational molecular modeling indicates that the mutation G77S onthe mature mutated protein involves the total unfolding of theC-terminal end of helix B caused by a steric hindrance with thephenylalanine residue at position 183 on helix D and by an unfavorableinteraction between an hydrophilic (serine at position 77) and anhydrophobic (leucine at position 35) amino acids on the loop betweenhelix A and helix B. The G77 residue is buried in the wild-type proteinstructure.

Thus, the mutated protein possesses a different three-dimensionalconformation from the natural wild-type EPO protein encoded by thewild-type EPO gene.

Computational molecular modeling also predicts that the presence of theamino acid serine at position 77 involves a significant modification ofthe structure and of the function of the natural wild-type EPO protein,notably by altering the affinity of the EPO for its receptor.

3/ Computational molecular modeling indicates that the mutation S120C onthe mature mutated protein involves a structural modification located onthe loop between helix C and helix D, in particular between the lysineat position 116 and the alanine at position 125. The hydrogen bondbetween S120 and K116 residues in the wild-type EPO protein structure isdisrupted in the mutated protein structure.

Thus, the mutated protein possesses a different three-dimensionalconformation from the natural wild-type EPO protein encoded by thewild-type EPO gene.

Computational molecular modeling also predicts that the presence of thecysteine amino acid at position 120 involves a significant modificationof the structure and of the function of the natural wild-type EPOprotein.

Other SNPs in conformity with the invention, namely: 465-486 (deletion),c577t, g602c, c1288t, c1347t, t1607c, g2228a, c2502t, g2634a, do notinvolve modification of the protein encoded by the nucleotide sequenceof the EPO gene at the level of the amino acid sequence SEQ ID NO. 2.

The SNPs c1288t, t1607c, g2634a are silent and the SNPs 465-486(deletion), c577t, g602c, c 1347t, g2228a, c2502t are non-coding.

Genotyping of the polynucleotides in conformity with the invention canbe carried out in such a fashion as to determine the allelic frequencyof these polynucleotides in a population. Two examples of genotyping aregiven, hereinafter in the experimental part, for the SNPs g1644a andc2621g.

The determination of the functionality of the polypeptides of theinvention can equally be carried out by a test of their biologicalactivity according to protocols described in the following publications:

-   -   Bittorf et al.; “Rapid activation of the MAP kinase pathway in        hematopoietic cells by erythropoietin, granulocyte-macrophage        colony-stimulating factor and interleukin-3”; Cell Signal; 1994;        March; 6(3): 305-11.    -   Chretien et al.; “Erythropoietin-induced erythroid        differentiation of the human erythroleukemia cell line TF-1        correlates with impaired STAT5 activation”; EMBO J.; 1996 August        15; 15(16): 4174-81.    -   Porteu et al.; “Functional regions of the mouse thrombopoietin        receptor cytoplasmic domain: evidence for a critical region        which is involved in differentiation and can be complemented by        erythropoietin”; Mol. Cell. Biol.; 1996 May; 16(5): 2473-82.    -   Pallard et al.; “Thrombopoietin activates a STAT5-like factor in        hematopoietic cells”; EMBO J.; 1995 June 15; 14(12): 2847-56.

The invention also has for an object the use of polynucleotides and ofpolypeptides in conformity with the invention as well as of therapeuticmolecules obtained and/or identified starting from these polynucleotidesand polypeptides, notably for the prevention and the treatment ofcertain human disorders and/or diseases.

Such molecules are particularly useful to prevent or to treat anemia, inparticular in patients under dialysis in renal insufficiency, as well asanemia resulting from chronic infections, inflammatory processes,radiotherapies, chemotherapies, as well as to prevent brain injury.

Such molecules are even more particularly useful to increase theproduction of autologous blood, notably in patients participating in adiffered autologous blood collection program to avoid the use of bloodfrom an other person.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A represents the modeling of the encoded protein according to theinvention comprising the SNP D70N and the natural wild-typeerythropoietin. FIG. 1B represents the modeling of the right part of themutated and wild-type proteins.

In FIGS. 1A and 1B, the black ribbon represents the structure of thenatural wild-type erythropoietin and the white ribbon represents thestructure of the mutated erythropoietin (D70N).

FIG. 2A represents the modeling of the encoded protein according to theinvention comprising the SNP G104S and the natural wild-typeerythropoietin. FIG. 2B represents the modeling of the inferior part ofthe mutated and wild-type proteins.

In FIGS. 2A and 2B the black ribbon represents the structure of thenatural wild-type erythropoietin and the white ribbon represents thestructure of the mutated erythropoietin (G104S).

FIG. 3A represents the modeling of the encoded protein according to theinvention comprising the SNP S 147C and the natural wild-typeerythropoietin. FIG. 3B represents the modeling of the upper left partof the mutated and wild-type proteins.

In FIGS. 3A and 3B the black ribbon represents the structure of thenatural wild-type erythropoietin and the white ribbon represents thestructure of the mutated erythropoietin (S147C).

FIG. 4 represents the effect of G104S mutated erythropoietin andwild-type erythropoietin (contained in protein extracts) onproliferation of cells from 32 D murine cell line stably transfectedwith human erythropoietin receptor. FIGS. 4A and 4B represent theresults from two independent experiments, respectively.

FIG. 5 represents the effect of purified G104S mutated erythropoietinand purified wild-type erythropoietin on proliferation of cells from 32D murine cell line stably transfected with human erythropoietinreceptor. FIGS. 5A and 5B represent the results from two independentexperiments, respectively.

FIG. 6 represents the erythroid colony formation after stimulation byG104S mutated erythropoietin (white triangles) or wild-typeerythropoietin (black squares).

FIG. 7 represents the binding capacity of G104S mutated erythropoietin(circles) and wild-type erythropoietin (stars) to the external part ofhuman EPO receptor. The data obtained with two concentrations oferythropoietin are represented: 7.5 nM in white, and 15 nM in black.

DETAILED DESCRIPTION OF THE INVENTION

Definitions.

“Nucleotide sequence of the reference wild-type gene” is understood asthe nucleotide sequence SEQ ID NO. 1 of the human EPO gene which isaccessible in GenBank under Accession number X02158 and described inJacobs K. et al.; “Isolation and characterization of genomic and cDNAclones of human erythropoietin”; Nature 313 (6005), 806-810 (1985).

“Natural wild-type erythropoietin protein” is understood as the matureprotein encoded by the nucleotide sequence of the reference wild-typeEPO gene. The natural wild-type immature EPO protein corresponds to thepeptide sequence SEQ ID NO. 2.

“Polynucleotide” is understood as a polyribonucleotide or apolydeoxyribonucleotide that can be a modified or non-modified DNA or anRNA.

The term polynucleotide includes, for example, a single strand or doublestrand DNA, a DNA composed of a mixture of one or several single strandregion(s) and of one or several double strand region(s), a single strandor double strand RNA and an RNA composed of a mixture of one or severalsingle strand region(s) and of one or several double strand region(s).The term polynucleotide can also include an RNA and/or a DNA includingone or several triple strand regions. Polynucleotide is equallyunderstood as the DNAs and RNAs containing one or several bases modifiedin such a fashion as to have a skeleton modified for reasons ofstability or for other reasons. By modified base is understood, forexample, the unusual bases such as inosine.

“Polypeptide” is understood as a peptide, an oligopeptide, an oligomeror a protein comprising at least two amino acids joined to each other bya normal or modified peptide bond, such as in the cases of the isostericpeptides, for example.

A polypeptide can be composed of amino acids other than the 20 aminoacids defined by the genetic code. A polypeptide can equally be composedof amino acids modified by natural processes, such as post-translationalmaturation processes or by chemical processes, which are well known to aperson skilled in the art. Such modifications are fully detailed in theliterature. These modifications can appear anywhere in the polypeptide:in the peptide skeleton, in the amino acid chain or even at the carboxy-or amino-terminal ends.

A polypeptide can be branched following an ubiquitination or be cyclicwith or without branching. This type of modification can be the resultof natural or synthetic post-translational processes that are well knownto a person skilled in the art.

For example, polypeptide modifications is understood to includeacetylation, acylation, ADP-ribosylation, amidation, covalent fixationof flavine, covalent fixation of heme, covalent fixation of a nucleotideor of a nucleotide derivative, covalent fixation of a lipid or of alipidic derivative, the covalent fixation of a phosphatidylinositol,covalent or non-covalent cross-linking, cyclization, disulfide bondformation, demethylation, cysteine formation, pyroglutamate formation,formylation, gamma-carboxylation, glycosylation including pegylation,GPI anchor formation, hydroxylation, iodization, methylation,myristoylation, oxidation, proteolytic processes, phosphorylation,prenylation, racemization, seneloylation, sulfatation, amino acidaddition such as arginylation or ubiquitination. Such modifications arefully detailed in the literature: PROTEINS-STRUCTURE AND MOLECULARPROPERTIES, 2^(nd) Ed., T. E. Creighton, N.Y., 1993, POST-TRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, 1983, Seifter et al. “Analysis for protein modifications andnonprotein cofactors”, Meth. Enzymol. (1990) 182: 626-646 et Rattan etal. “Protein Synthesis: Post-translational Modifications and Aging”, AnnNY Acad Sci (1992) 663: 48-62.

A “hyperglycosylated polypeptide” or “hyperglycosylated analog of apolypeptide” is understood as a polypeptide whose amino acid sequencehas been altered in such a way as to possess at least one moreadditional glycosylation site or a polypeptide with the same amino acidsequence but whose glycosylation level has been increased.

“Isolated polynucleotide” or “isolated polypeptide” is understood as apolynucleotide or a polypeptide such as previously defined which isisolated from the human body or otherwise produced by a technicalprocess.

“Identity” is understood as the measurement of nucleotide or polypeptidesequences identity. Identity is a term well known to a person skilled inthe art and well described in the literature. See COMPUTATIONALMOLECULAR BIOLOGY, Lesk, A. M., Ed., Oxford University Press, New York,1998; BIOCOMPUTING INFORMATICS AND GENOME PROJECT, Smith, D. W., Ed.,Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PARTI, Griffin, A. M. and Griffin H. G., Ed, Humana Press, New Jersey, 1994;et SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., AcademicPress, 1987.

The methods commonly employed to determine the identity and thesimilarity between two sequences are equally well described in theliterature. See GUIDE TO HUGE COMPUTER, Martin J. Bishop, Ed, AcademicPress, San Diego, 1994, and Carillo H. and Lipton D., Siam J AppliedMath (1988) 48: 1073.

A polynucleotide having, for example, an identity of at least 95 % withthe nucleotide sequence SEQ ID NO. 1 is a polynucleotide which containsat most 5 points of mutation over 100 nucleotides, compared to saidsequence.

These points of mutation can be one (or several) substitution(s),addition(s) and/or deletion(s) of one (or several) nucleotide(s).

In the same way, a polypeptide having, for example, an identity of atleast 95 % with the amino acid sequence SEQ ID NO. 2 is a polypeptidethat contains at most 5 points of mutation over 100 amino acids,compared to said sequence.

These points of mutation can be one (or several) substitution(s),addition(s) and/or deletion(s) of one (or several) amino acid(s).

The polynucleotides and the polypeptides according to the inventionwhich are not totally identical with respectively the nucleotidesequence SEQ ID NO. 1 or the amino acid sequence SEQ ID NO. 2, it beingunderstood that these sequences contains at least one of the SNPs of theinvention, are considered as variants of these sequences.

Usually a polynucleotide according to the invention possesses the sameor practically the same biological activity as the nucleotide sequenceSEQ ID NO. 1 comprising at least one of the SNPs of the invention.

Similarly, usually a polypeptide according to the invention possessesthe same or practically the same biological activity as the amino acidsequence SEQ ID NO. 2 comprising at least one of the coding SNPs of theinvention.

A variant, according to the invention, can be obtained, for example, bysite-directed mutagenesis or by direct synthesis.

“SNP” is understood as any natural variation of a base in a nucleotidesequence. A SNP on a nucleotide sequence can be coding, silent ornon-coding.

A coding SNP is a polymorphism included in the coding sequence of anucleotide sequence that involves a modification of an amino acid in thesequence of amino acids encoded by this nucleotide sequence. In thiscase, the term SNP applies equally, by extension, to a mutation in anamino acid sequence.

A silent SNP is a polymorphism included in the coding sequence of anucleotide sequence that does not involve a modification of an aminoacid in the amino acid sequence encoded by this nucleotide sequence.

A non-coding SNP is a polymorphism included in the non-coding sequenceof a nucleotide sequence. This polymorphism can notably be found in anintron, a splicing zone, a transcription promoter or an enhancer sitesequence.

“Functional SNP” is understood as a SNP, such as previously defined,which is included in a nucleotide sequence or an amino acid sequence,having a functionality.

“Functionality” is understood as the biological activity of apolypeptide or of a polynucleotide.

The functionality of a polypeptide or of a polynucleotide according tothe invention can consist in a conservation, an augmentation, areduction or a suppression of the biological activity of the polypeptideencoded by the nucleotide sequence of the wild-type reference gene or ofthis latter nucleotide sequence.

The functionality of a polypeptide or of a polynucleotide according tothe invention can equally consist in a change in the nature of thebiological activity of the polypeptide encoded by the nucleotidesequence of the reference wild-type gene or of this latter nucleotidesequence.

The biological activity can, notably, be linked to the affinity or tothe absence of affinity of a polypeptide according to the invention witha receptor.

Polynucleotides.

The present invention has for its first object an isolatedpolynucleotide comprising:

-   -   a) a nucleotide sequence having at least 80% identity,        preferably at least 90% identity, more preferably at least 95%        identity and still more preferably at least 99% identity with        the sequence SEQ ID NO. 1 or its coding sequence (of the        nucleotide 615 to the nucleotide 2763), it being understood that        this nucleotide sequence comprises at least one of the following        coding SNPs: g1644a, g2357a, c2621g, or    -   b) a nucleotide sequence complementary to a nucleotide sequence        under a).

The present invention relates equally to an isolated polynucleotidecomprising:

-   -   a) the nucleotide sequence SEQ ID NO. 1 or its coding sequence,        it being understood that each of these sequences comprises at        least one of the following coding SNPs: g1644a, g2357a, c2621g,        or    -   b) a nucleotide sequence complementary to a nucleotide sequence        under a).

Preferably, the polynucleotide of the invention consists of the sequenceSEQ ID NO. 1 or its coding sequence, it being understood that each ofthese sequences comprises at least one of the following coding SNPs:g1644a, g2357a, c2621g.

According to the invention, the polynucleotide previously definedcomprises a single coding SNP selected from the group consisting of:g1644a, g2357a, and c2621g.

A polynucleotide such as previously defined can equally include at leastone of the following non-coding and silent SNPs: 465-486 (deletion),c577t, g602c, c1288t, c1347t, t1607c, g2228a, c2502t, g2634a.

The present invention equally has for its object an isolatedpolynucleotide comprising or consisting of:

-   -   a) the nucleotide sequence SEQ ID NO. 1 or if necessary its        coding sequence, it being understood that each of these        sequences comprises at least one of the following non coding or        silent SNPs: 465-486 (deletion), c577t, g602c, c1288t, c1347t,        t1607c, g2228a, c2502t, g2634a, or    -   b) a nucleotide sequence complementary to a nucleotide sequence        under a). It is understood that the following silent SNPs        c1288t, t1607c, g2634a, are located in the coding sequence of        the nucleotide sequence SEQ ID NO. 1.

The present invention concerns also an isolated polynucleotideconsisting of a part of:

-   -   a) a nucleotide sequence SEQ ID NO. 1 or if necessary its coding        sequence, it being understood that each of these sequences        comprises at least one of the following SNPs: 465 486        (deletion), c577t, g602c, c1288t, c1347t, t1607c, g1644a,        g2228a, g2357a, c2502c2621g, g2634a, or    -   b) a nucleotide sequence complementary to a nucleotide sequence        under a). said isolated polynucleotide being composed of at        least 10 nucleotides.

The present invention also has for its object an isolated polynucleotidecoding for a polypeptide comprising:

-   -   a) the amino acid sequence SEQ ID NO. 2, or    -   b) the amino acid sequence comprising the amino acids included        between positions 28 and 193 of the sequence of amino acids SEQ        ID NO. 2, it being understood that each of the amino acid        sequences under a) and b) comprises at least one of the        following coding SNPs: D70N, G104S, S 147C.        It is understood, in the sense of the present invention, that        the numbering corresponding to the positioning of the D70N,        G104S, S147C SNPs is relative to the numbering of the amino acid        sequence SEQ ID NO. 2.

According to a preferred object of the invention, the previously definedpolypeptide comprises a single coding SNP such as defined above.

More preferably, the present invention also has for its object anisolated polynucleotide coding for a polypeptide comprising all or partof the amino acid sequence SEQ ID NO. 2 and having SNP G104S.

Preferably a polynucleotide according to the invention is composed of aDNA or RNA molecule.

A polynucleotide according to the invention can be obtained by standardDNA or RNA synthetic methods.

A polynucleotide according to the invention can equally be obtained bysite-directed mutagenesis starting from the nucleotide sequence of theEPO gene by modifying the wild-type nucleotide by the mutated nucleotidefor each SNP on the nucleotide sequence SEQ ID NO. 1.

For example, a polynucleotide according to the invention, comprising aSNP g2357a can be obtained by site-directed mutagenesis starting fromthe nucleotide sequence of the EPO gene by modifying the nucleotide g bythe nucleotide a at position 2357 on the nucleotide sequence SEQ ID NO.1.

The processes of site-directed mutagenesis that can be implemented inthis way are well known to a person skilled in the art. The publicationof TA Kunkel in 1985 in “Proc. Natl. Acad. Sci. USA” 82:488 can notablybe mentioned.

An isolated polynucleotide can equally include, for example, nucleotidesequences coding for pre-, pro- or pre-pro-protein amino acid sequencesor marker amino acid sequences, such as hexa-histidine peptide.

A polynucleotide of the invention can equally be associated withnucleotide sequences coding for other proteins or protein fragments inorder to obtain fusion proteins or other purification products.

A polynucleotide according to the invention can equally includenucleotide sequences such as the 5′ and/or 3′ non-coding sequences, suchas, for example, transcribed or non-transcribed sequences, translated ornon-translated sequences, splicing signal sequences, polyadenylatedsequences, ribosome binding sequences or even sequences which stabilizemRNA.

A nucleotide sequence complementary to the nucleotide or polynucleotidesequence is defined as one that can hybridize with this nucleotidesequence, under stringent conditions.

By “stringent hybridization conditions” is generally but not necessarilyunderstood the chemical conditions that permit a hybridization when thenucleotide sequences have an identity of at least 80%, preferablygreater than or equal to 90%, still more preferably greater than orequal to 95% and most preferably greater than or equal to 97%.

The stringent conditions can be obtained according to methods well knownto a person skilled in the art and, for example, by an incubation of thepolynucleotides, at 42° C., in a solution comprising 50% formamide,5×SSC (150 mM of NaCI, 15 mM of trisodium citrate), 50 mM of sodiumphosphate (pH=7.6), 5× Denhardt Solution, 10% dextran sulfate and 20 μgdenatured salmon sperm DNA, followed by washing the filters at 0.1×SSC,at 65° C.

Within the scope of the invention, when the stringent hybridizationconditions permit hybridization of the nucleotide sequences having anidentity equal to 100%, the nucleotide sequence is considered to bestrictly complementary to the nucleotide sequence such as describedunder a).

It is understood within the meaning of the present invention that thenucleotide sequence complementary to a nucleotide sequence comprises atleast one anti-sense SNP according to the invention.

Thus, for example, if the nucleotide sequence comprises the SNP g1644a,its complementary nucleotide sequence comprises the t nucleotide atequivalent of position 1644.

Identification, Hybridization and/or Amplification of a PolynucleotideComprising a SNP

The present invention also has for its object the use of all or part ofa previously defined polynucleotide, in order to identify, hybridizeand/or amplify all or part of a polynucleotide consisting of thenucleotide sequence SEQ ID NO. 1 or if necessary its coding sequence (ofthe nucleotide 615 to the nucleotide 2763), it being understood thateach one of these sequences comprises at least one of the followingSNPs: 465-486 (deletion), c577t, g602c, c1288t, c1347t, t1607c, g1644a,g2228a, g2357a, c2502t, c2621, g2634a.

Genotyping and Determination of the Frequency of a SNP

The present invention equally has for its object the use of all or partof a polynucleotide according to the invention for the genotyping of anucleotide sequence which has 90 to 100% identity with the nucleotidesequence of EPO gene and which comprises at least one of the followingSNPs: 465-486 (deletion), c577t, g602c, c1288t, c1347t, t1607c, g1644a,g2228a, g2357a, c2502t, c2621g, g2634a.

According to the invention, the genotyping may be carried out on anindividual or a population of individuals.

Within the meaning of the invention, genotyping is defined as a processfor the determination of the genotype of an individual or of apopulation of individuals. Genotype consists of the alleles present atone or more specific loci. “Population of individuals” is understood asa group of determined individuals selected in random or non-randomfashion. These individuals can be humans, animals, microorganisms orplants.

Usually, the group of individuals comprises at least 10 persons,preferably from 100 to 300 persons.

The individuals can be selected according to their ethnicity oraccording to their phenotype, notably those who are affected by thefollowing disorders and/or diseases: cancers and tumors, infectiousdiseases, venereal diseases, immunologically related diseases and/orautoimmune diseases and disorders, cardiovascular diseases, metabolicdiseases, central nervous system diseases, gastrointestinal disorders,and disorders connected with chemotherapy treatments.

Said cancers and tumors include carcinomas comprising metastasizingrenal carcinomas, melanomas, lymphomas comprising follicular lymphomasand cutaneous T cell lymphoma, leukemias comprising chronic lymphocyticleukemia and chronic myeloid leukemia, cancers of the liver, neck, headand kidneys, multiple myelomas, carcinoid tumors and tumors that appearfollowing an immune deficiency comprising Kaposi's sarcoma in the caseof AIDS.

Said infectious diseases include viral infections comprising chronichepatitis B and C and HIV/AIDS, infectious pneumonias, and venerealdiseases, such as genital warts. Said immunologically andauto-immunologically related diseases may include the rejection oftissue or organ grafts, allergies, asthma, psoriasis, rheumatoidarthritis, multiple sclerosis, Crohn's disease and ulcerative colitis.

Said cardiovascular diseases may include brain injury and anemiasincluding anemia in patients under dialysis in renal insufficiency, aswell as anemia resulting from chronic infections, inflammatoryprocesses, radiotherapies, and chemotherapies.

Said metabolic diseases may include such non-immune associated diseasesas obesity.

Said diseases of the central nervous system may include Alzheimer'sdisease, Parkinson's disease, schizophrenia and depression.

Said diseases and disorders may also include wound healing andosteoporosis.

The compounds of the invention may preferably be used for thepreparation of a therapeutic compound intended to increase theproduction of autologous blood, notably in patients participating in adiffered autologous blood collection program to avoid the use of bloodfrom an other person.

A functional SNP according to the invention is preferably genotyped in apopulation of individuals.

Many technologies exist which can be implemented in order to genotypeSNPs (see notably Kwok Pharmacogenomics, 2000, vol 1, pp 95-100.“High-throughput genotyping assay approaches”). These technologies arebased on one of the four following principles: allele specificoligonucleotide hybridization, oligonucleotide elongation bydideoxynucleotides optionally in the presence of deoxynucleotides,ligation of allele specific oligonucleotides or cleavage of allelespecific oligonucleotides. Each of these technologies can be coupled toa detection system such as measurement of direct or polarizedfluorescence, or mass spectrometry.

Genotyping can notably be carried out by minisequencing with hot ddNTPs(2 different ddNTPs labeled by different fluorophores) and cold ddNTPs(2 different non labeled ddNTPs), in connection with a polarizedfluorescence scanner. The minisequencing protocol with reading ofpolarized fluorescence (FP-TDI Technology or Fluorescence PolarizationTemplate-direct Dye-Terminator Incorporation) is well known to a personskilled in the art.

It can be carried out on a product obtained after amplification bypolymerase chain reaction (PCR) of the DNA of each individual. This PCRproduct is selected to cover the polynucleotide genic region containingthe studied SNP. After the last step in the PCR thermocycler, the plateis placed on a polarized fluorescence scanner for a reading of thelabeled bases by using fluorophore specific excitation and emissionfilters. The intensity values of the labeled bases are reported on agraph.

For the PCR amplification, in the case of a SNP of the invention, thesense and antisense primers, respectively, can easily be selected by aperson skilled in the art according to the position of the SNPs of theinvention.

For example, the sense and antisense nucleotide sequences for the PCRamplification of a fragment whose sequence comprises the SNPs g2228a,g2357a, c2502t, c2621 g and/or g2634a can be, respectively:

-   SEQ ID NO. 3: Sense primer (A): TTGCATACCTTCTGTTTGCT-   SEQ ID NO. 4: Antisense primer (B): CACAAGCAATGTTGGTGAG

These nucleotide sequences permit amplification of a fragment having alength of 626 nucleotides, of the nucleotide 2192 to the nucleotide 2817in the nucleotide sequence SEQ ID NO. 1.

A statistical analysis of the frequency of each allele (allelicfrequency) encoded by the gene comprising the SNP in the population ofindividuals is then achieved, which permits determination of theimportance of their impact and their distribution in the differentsub-groups and notably, if necessary, the diverse ethnic groups thatconstitute this population of individuals.

The genotyping data are analyzed in order to estimate the distributionfrequency of the different alleles observed in the studied populations.The calculations of the allelic frequencies can be carried out with thehelp of software such as SAS-suite® (SAS) or SPLUS® (MathSoft). Thecomparison of the allelic distributions of a SNP of the invention acrossdifferent ethnic groups of the population of individuals can be carriedout by means of the software ARLEQUIN® and SAS-suite®.

The present invention also concerns the use of a polynucleotideaccording to the invention for the research of one variation in the EPOnucleotide sequence in one individual.

SNPs of the Invention as Genetic Markers

Whereas SNPs modifying functional sequences of genes (e.g. promoter,splicing sites, coding region) are likely to be directly related todisease susceptibility or resistance, all SNPs (functional or not) mayprovide valuable markers for the identification of one or several genesinvolved in these disease states and, consequently, may be indirectlyrelated to these disease states (See Cargill et al. (1999). NatureGenetics 22:231-238; Riley et al. (2000). Pharmacogenomics 1:39-47;Roberts L. (2000). Science 287: 1898-1899).

Thus, the present invention also concerns a databank comprising at leastone of the following SNPs: 465-486 (deletion), c577t, g602c, c1288t,c1347t, t1607c, g1644a, g2228a, g2357a, c2502t, c2621g, g2634a, in apolynucleotide of the EPO gene.

It is understood that said SNPs are numbered in accordance with thenucleotide sequence SEQ ID NO. 1.

This databank may be analyzed for determining statistically relevantassociations between:

-   (i) at least one of the following SNPs: 465-486 (deletion), c577t,    g602c, c1288t, c1347t, t1607c, g1644a, g2228a, g2357a, c2502t,    c2621g, g2634a, in a polynucleotide of the EPO gene, and-   (ii) a disease or a resistance to a disease.

The present invention also concerns the use of at least one of thefollowing SNPs: 465-486 (deletion), c577t, g602c, c1288t, c1347t,t1607c, g1644a, g2228a, g2357a, c2502t, c2621g, g2634a, in apolynucleotide of the EPO gene, for developing diagnostic/prognostickits for a disease or a resistance to a disease.

A SNP of the invention such as defined above may be directly orindirectly associated to a disease or a resistance to a disease.

Preferably, these diseases may be those which are defined as mentionedabove.

Expression Vector and Host Cell

The present invention also has for its object a recombinant vectorcomprising at least one polynucleotide according to the invention.

Numerous expression systems can be used like, for example, chromosomes,episomes, derived viruses. More particularly, the recombinant vectorsused can be derived from bacterial plasmids, transposons, yeast episome,insertion elements, yeast chromosome elements, viruses such asbaculovirus, papilloma viruses such as SV40, vaccinia viruses,adenoviruses, fox pox viruses, pseudorabies viruses, retroviruses.

These recombinant vectors can equally be cosmid or phagemid derivatives.The nucleotide sequence can be inserted in the recombinant expressionvector by methods well known to a person skilled in the art such as, forexample, those that are described in MOLECULAR CLONING: A LABORATORYMANUAL, Sambrook et al., 2^(nd) Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989).

The recombinant vector can include nucleotide sequences that control theregulation of the polynucleotide expression as well as nucleotidesequences permitting the expression and the transcription of apolynucleotide of the invention and the translation of a polypeptide ofthe invention, these sequences being selected according to the hostcells that are used.

Thus, for example, an appropriate secretion signal can be integrated inthe recombinant vector so that the polypeptide, encoded by thepolynucleotide of the invention, will be directed towards the lumen ofthe endoplasmic reticulum, towards the periplasmic space, on themembrane or towards the extracellular environment.

The present invention also has for its object a host cell comprising arecombinant vector according to the invention.

The introduction of the recombinant vector in a host cell can be carriedout according to methods that are well known to a person skilled in theart such as those described in BASIC METHODS IN MOLECULAR BIOLOGY, Daviset al., 1986 and MOLECULAR CLONING: A LABORATORY MANUAL, supra, such astransfection by calcium phosphate, transfection by DEAE dextran,transfection, microinjection, transfection by cationic lipids,electroporation, transduction or infection.

The host cell can be, for example, bacterial cells such as cells ofstreptococci, staphylococci, E. coli or Bacillus subtilis, cells offungi such as yeast cells and cells of Aspergillus, Streptoniyces,insect cells such as cells of Drosophilia S2 and of Spodoptera Sf9,animal cells, such as CHO, COS, HeLa, C127, BHK, HEK 293 cells and humancells of the subject to treat or even plant cells.

The host cells can be used, for example, to express a polypeptide of theinvention or as active product in pharmaceutical compositions, as willbe seen hereinafter.

Polypeptides.

The present invention also has for its object an isolated polypeptidecomprising an amino acid sequence having at least 80% identity,preferably at least 90% identity, more preferably at least 95% identityand still more preferably at least 99% identity with:

-   -   a) the amino acid sequence SEQ ID NO. 2 or with    -   b) the amino acid sequence comprising the amino acids included        between positions 28 and 193 of the amino acid sequence SEQ ID        NO. 2,        it being understood that each of the amino acid sequences        under a) and b) contains at least one of the following coding        SNPs: D70N, G104S, S147C.

The polypeptide of the invention can equally comprise:

-   -   a) the amino acid sequence SEQ ID NO. 2, or    -   b) the amino acid sequence containing the amino acids included        between positions 28 and 193 of the amino acid sequence SEQ ID        NO. 2,        it being understood that each of the amino acid sequences        under a) and b) contains at least one of the following coding        SNPs: D70N, G104S, S147C.

The polypeptide of the invention can more particularly consist of:

-   -   a) the amino acid sequence SEQ ID NO. 2, or    -   b) the amino acid sequence containing the amino acids included        between positions 28 and 193 of the amino acid sequence SEQ ID        NO. 2,        it being understood that each of the amino acid sequences        under a) and b) contains at least one of the following coding        SNys: D70N, G104S, S 147C.

Preferably, a polypeptide according to the invention contains a singlecoding SNP selected from the group consisting of: D70N, G104S, andS147C.

More preferably, a polypeptide according to the invention comprisesamino acids 28 through 193 of the amino acid sequence SEQ ID NO. 2 andhas SNP G104S.

The present invention also concerns a hyperglycosylated analog of apolypeptide according to the invention in order to improve itstherapeutic properties.

Preferably, the present invention concerns hyperglycosylated analogs ofa polypeptide comprising amino acids 28 through 193 of the amino acidsequence SEQ ID NO. 2 and having SNP G104S.

More preferably, the present invention concerns pegylated analogs of apolypeptide comprising amino acids 28 through 193 of the amino acidsequence SEQ ID NO. 2, and having SNP G104S.

Indeed, it is known in the art that the oligosaccharide component cansignificantly affect properties relevant to efficacy of a therapeuticglycoprotein, including physical stability, resistance to proteaseattack, interactions with the immune system, pharmacokinetics andspecific biological activity (See, for example, Dube et al. J. Biol.Chem. 263, 17516 (1988); Delorme et al. Biochemistry 31, 9871-9876(1992)). Whereas human wild-type urinary derived EPO and recombinantwild-type human EPO contain three N-linked and one O-linkedoligosaccharide chains, which together comprise about 40% of the totalmolecular weight of the glycoprotein, it is still possible to increasethe number of carbohydrate chains on the protein. Techniques that permitthe increase in the number of carbohydrate chains on a protein are wellknown by the one skilled in the art, including the following:

-   -   introduction of new sites available for glycosylation using        site-directed mutagenesis creating amino acid residue        substitution or addition (see EP0640619 and U.S. patent        application Ser. No. 09/853731, published as Publication No.        20020037841, for example).    -   glycosylation engineering of proteins by using a host cell which        harbor the nucleic acid encoding the protein of interest and at        least one nucleic acid encoding a glycoprotein-modifying        glycosyl transferase as suggested by WO9954342 application.

The present invention equally has for its object a process for thepreparation of the above-described polypeptide, in which a previouslydefined host cell is cultivated in a culture medium and said polypeptideis isolated from the culture medium.

The polypeptide can be purified from the host cells' culture medium,according to methods well known to a person skilled in the art such asprecipitation with chaotropic agents such as salts, in particularammonium sulfate, ethanol, acetone or trichloroacetic acid; acidextraction; ion exchange chromatography; phosphocellulosechromatography; hydrophobic interaction chromatography; affinitychromatography; hydroxyapatite chromatography or exclusionchromatographies.

“Culture medium” is understood as the medium in which the polypeptide ofthe invention is isolated or purified. This medium can be composed ofthe extracellular medium and/or the cellular lysate. Techniques wellknown to a person skilled in the art equally permit him or her to giveback an active conformation to the polypeptide, if the conformation ofsaid polypeptide was altered during the isolation or the purification.

Antibodies.

The present invention also has for its object a process for obtaining animmunospecific antibody.

“Antibody” is understood as including monoclonal, polyclonal, chimeric,simple chain, humanized antibodies as well as the Fab fragments,including Fab or immunoglobulin expression library products.

An immunospecific antibody can be obtained by immunization of an animalwith a polypeptide according to the invention.

The invention also relates to an immunospecific antibody for apolypeptide according to the invention, such as defined previously.

A polypeptide according to the invention, one of its fragments, ananalog, one of its variants or a cell expressing this polypeptide canalso be used to produce immunospecific antibodies.

The term “immunospecific” means that the antibody possesses a betteraffinity for the polypeptide of the invention than for otherpolypeptides known in the prior art.

The immunospecific antibodies can be obtained by administration of apolypeptide of the invention, of one of its fragments, of an analog orof an epitopic fragment or of a cell expressing this polynucleotide in amammal, preferably non human, according to methods well known to aperson skilled in the art.

For the preparation of monoclonal antibodies, typical methods forantibody production can be used, starting from cell lines, such as thehybridoma technique (Kohler et al., Nature (1975) 256: 495-497), thetrioma technique, the human B cell hybridoma technique (Kozbor et al.,Immunology Today (1983) 4: 72) and the EBV hybridoma technique (Cole etal., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss,1985).

The techniques of single chain antibody production such as described,for example, in U.S. Pat. No. 4,946,778 can equally be used.

Transgenic animals such as mice, for example, can equally be used toproduce humanized antibodies.

Agents Interacting with the Polypeptide of the Invention

The present invention equally has for its object a process for theidentification of an agent activating or inhibiting a polypeptideaccording to the invention, comprising:

-   -   a) the preparation of a recombinant vector comprising a        polynucleotide according to the invention containing at least        one coding SNP,    -   b) the preparation of host cells comprising a recombinant vector        according to a),    -   c) the contacting of host cells according to b) with an agent to        be tested, and    -   d) the determination of the activating or inhibiting effect        generated by the agent to test.

A polypeptide according to the invention can also be employed for aprocess for screening compounds that interact with it.

These compounds can be activating (agonists) or inhibiting (antagonists)agents of intrinsic activity of a polypeptide according to theinvention. These compounds can equally be ligands or substrates of apolypeptide of the invention. See Coligan et al., Current Protocols inImmunology 1 (2), Chapter 5 (1991).

In general, in order to implement such a process, it is first desirableto produce appropriate host cells that express a polypeptide accordingto the invention. Such cells can be, for example, cells of mammals,yeasts, insects such as Drosophilia or bacteria such as E. coli.

These cells or membrane extracts of these cells are then placed in thepresence of compounds to be tested.

The binding capacity of the compounds to be tested with the polypeptideof the invention can then be observed, as well as the inhibition or theactivation of the functional response.

Step d) of the above process can be implemented by using an agent to betested that is directly or indirectly labeled. It can also include acompetition test, by using a labeled or non-labeled agent and a labeledcompetitor agent.

It can equally be determined if an agent to be tested generates anactivation or inhibition signal on cells expressing the polypeptide ofthe invention by using detection means appropriately chosen according tothe signal to be detected.

Such activating or inhibiting agents can be polynucleotides, and incertain cases oligonucleotides or polypeptides, such as proteins orantibodies, for example.

The present invention also has for its object a process for theidentification of an agent activated or inhibited by a polypeptideaccording to the invention, comprising:

-   -   a) the preparation of a recombinant vector comprising a        polynucleotide according to the invention containing at least        one coding SNP,    -   b) the preparation of host cells comprising a recombinant vector        according to a),    -   c) placing the host cells according to b) in the presence of an        agent to be tested, and    -   d) the determination of the activating or inhibiting effect        generated by the polypeptide on the agent to be tested.

An agent activated or inhibited by the polypeptide of the invention isan agent that responds, respectively, by an activation or an inhibitionin the presence of this polypeptide. The agents, activated or inhibiteddirectly or indirectly by the polypeptide of the invention, can consistof polypeptides such as, for example, membranal or nuclear receptors,kinases and more preferably tyrosine kinases, transcription factor orpolynucleotides.

Detection of Diseases

The present invention also has for object a process for analyzing thebiological characteristics of a polynucleotide according to theinvention and/or of a polypeptide according to the invention in asubject, comprising at least one of the following:

-   -   a) Determining the presence or the absence of a polynucleotide        according to the invention in the genome of a subject;    -   b) Determining the level of expression of a polynucleotide        according to the invention in a subject;    -   c) Determining the presence or the absence of a polypeptide        according to the invention in a subject;    -   d) Determining the concentration of a polypeptide according to        the invention in a subject; and/or    -   e) Determining the functionality of a polypeptide according to        the invention in a subject.

These biological characteristics may be analyzed in a subject or in asample from a subject.

These biological characteristics may permit genetic diagnosis and/ordetermination of whether a subject is affected or at risk of beingaffected or, to the contrary, presents a partial resistance to thedevelopment of a disease, an indisposition or a disorder linked to thepresence of a polynucleotide according to the invention and/or apolypeptide according to the invention. These diseases can be disordersand/or human diseases, such as cancers and tumors, infectious diseases,venereal diseases, immunologically related diseases and/or autoimmunediseases and disorders, cardiovascular diseases, metabolic diseases,central nervous system diseases, gastrointestinal disorders, anddisorders connected with chemotherapy treatments.

Said cancers and tumors include carcinomas comprising metastasizingrenal carcinomas, melanomas, lymphomas comprising follicular lymphomasand cutaneous T cell lymphoma, leukemias comprising chronic lymphocyticleukemia and chronic myeloid leukemia, cancers of the liver, neck, headand kidneys, multiple myelomas, carcinoid tumors and tumors that appearfollowing an immune deficiency comprising Kaposi's sarcoma in the caseof AIDS.

Said infectious diseases include viral infections comprising chronichepatitis B and C and HIV/AIDS, infectious pneumonias, and venerealdiseases, such as genital warts.

Said immunologically and auto-immunologically related diseases mayinclude the rejection of tissue or organ grafts, allergies, asthma,psoriasis, rheumatoid arthritis, multiple sclerosis, Crohn's disease andulcerative colitis.

Said cardiovascular diseases may include brain injury and anemiasincluding anemia in patients under dialysis in renal insufficiency, aswell as anemia resulting from chronic infections, inflammatoryprocesses, radiotherapies, and chemotherapies.

Said metabolic diseases may include such non-immune associated diseasesas obesity.

Said diseases of the central nervous system may include Alzheimer'sdisease, Parkinson's disease, schizophrenia and depression.

Said diseases and disorders may also include wound healing andosteoporosis.

This process also permits genetic diagnosis of a disease or resistanceto a disease linked to the presence, in a subject, of the mutant alleleencoded by a SNP according to the invention.

Preferably, in step a), the presence or absence of a polynucleotide,containing at least one coding SNP such as previously defined, is goingto be detected.

The detection of the polynucleotide may be carried out starting frombiological samples from the subject to be studied, such as cells, blood,urine, saliva, or starting from a biopsy or an autopsy of the subject tobe studied. The genomic DNA may be used for the detection directly orafter a PCR amplification, for example. RNA or cDNA can equally be usedin a similar fashion.

It is then possible to compare the nucleotide sequence of apolynucleotide according to the invention with the nucleotide sequencedetected in the genome of the subject.

The comparison of the nucleotide sequences can be carried out bysequencing, by DNA hybridization methods, by mobility difference of theDNA fragments on an electrophoresis gel with or without denaturingagents or by melting temperature difference.

See Myers et al., Science (1985) 230: 1242. Such modifications in thestructure of the nucleotide sequence at a precise point can equally berevealed by nuclease protection tests, such as RNase and the S1 nucleaseor also by chemical cleaving agents. See Cotton et al., Proc. Nat. Acad.Sci. USA (1985) 85: 4397-4401. Oligonucleotide probes comprising apolynucleotide fragment of the invention can equally be used to conductthe screening.

Many methods well known to a person skilled in the art can be used todetermine the expression of a polynucleotide of the invention and toidentify the genetic variability of this polynucleotide (See Chee etal., Science (1996), Vol 274, pp 610-613).

In step b), the level of expression of the polynucleotide may bemeasured by quantifying the level of RNA encoded by this polynucleotide(and coding for a polypeptide) according to methods well known to aperson skilled in the art as, for example, by PCR, RT-PCR, RNaseprotection, Northern blot, and other hybridization methods.

In step c) and d) the presence or the absence as well as theconcentration of a polypeptide according to the invention in a subjector a sample from a subject may be carried out by well known methods suchas, for example, by radioimmunoassay, competitive binding tests, Westernblot and ELISA tests.

Consecutively to step d), the determined concentration of thepolypeptide according to the invention can be compared with the naturalwild-type EPO protein concentration usually found in a subject.

A person skilled in the art can identify the threshold above or belowwhich appears the sensitivity or, to the contrary, the resistance to thedisease, the indisposition or the disorder evoked above, with the helpof prior art publications or by conventional tests or assays, such asthose that are previously mentioned.

In step e), the determination of the functionality of a polypeptideaccording to the invention may be carried out by methods well known to aperson skilled in the art as, for example, by in vitro tests such asabove mentioned or by an use of host cells expressing said polypeptide.

Therapeutic Compounds and Treatments of Diseases

The present invention also has for its object a therapeutic compoundcontaining, by way of active agent, a polypeptide according to theinvention and/or a hyperglycosylated analog of the polypeptidecomprising amino acids 28 through 193 of the amino acid sequence SEQ IDNO. 2 and having SNP G104S.

The invention also relates to the use of a polypeptide according to theinvention and/or a hyperglycosylated analog of the polypeptidecomprising amino acids 28 through 193 of the amino acid sequence SEQ IDNO. 2 and having SNP G104S, for the manufacture of a therapeuticcompound intended for the prevention or the treatment of different humandisorders and/or diseases. These diseases can be disorders and/or humandiseases, such as cancers and tumors, infectious diseases, venerealdiseases, immunologically related diseases and/or autoimmune diseasesand disorders, cardiovascular diseases, metabolic diseases, centralnervous system diseases, gastrointestinal disorders, and disordersconnected with chemotherapy treatments.

Said cancers and tumors include carcinomas comprising metastasizingrenal carcinomas, melanomas, lymphomas comprising follicular lymphomasand cutaneous T cell lymphoma, leukemias comprising chronic lymphocyticleukemia and chronic myeloid leukemia, cancers of the liver, neck, headand kidneys, multiple myelomas, carcinoid tumors and tumors that appearfollowing an immune deficiency comprising Kaposi's sarcoma in the caseof AIDS.

Said infectious diseases include viral infections comprising chronichepatitis B and C and HIV/AIDS, infectious pneumonias, and venerealdiseases, such as genital warts.

Said immunologically and auto-immunologically related diseases mayinclude the rejection of tissue or organ grafts, allergies, asthma,psoriasis, rheumatoid arthritis, multiple sclerosis, Crohn's disease andulcerative colitis.

Said cardiovascular diseases may include brain injury and anemiasincluding anemia in patients under dialysis in renal insufficiency, aswell as anemia resulting from chronic infections, inflammatoryprocesses, radiotherapies, and chemotherapies.

Said metabolic disease may include such non-immune associated diseasesas obesity.

Said diseases of the central nervous system may include Alzheimer'sdisease, Parkinson's disease, schizophrenia and depression.

Said diseases and disorders may also include wound healing andosteoporosis.

The compounds of the invention may preferably be used for thepreparation of a therapeutic compound intended to increase theproduction of autologous blood, notably in patients participating in adiffered autologous blood collection program to avoid the use of bloodfrom an other person.

Preferably, a polypeptide according to the invention and/or ahyperglycosylated analog of the polypeptide comprising amino acids 28through 193 of the amino acid sequence SEQ ID NO. 2 and having SNP G104Scan also be used for the manufacture of a therapeutic compound intended:

-   -   to prevent or treat anemia, in particular in patients under        dialysis in renal insufficiency, as well as anemia resulting        from chronic infections, inflammatory processes, radiotherapies,        chemotherapies, and/or    -   to increase the production of autologous blood, notably in        patients participating in a differed autologous blood collection        program to avoid the use of blood from an other person, and/or    -   to prevent brain injury.

Certain of the compounds permitting to obtain the polypeptide accordingto the invention as well as the compounds obtained or identified by orfrom this polypeptide can likewise be used for the therapeutic treatmentof the human body, i.e. as a therapeutic compound.

This is why the present invention also has for an object a therapeuticcompound containing, by way of active agent, a polynucleotide accordingto the invention containing at least one previously defined SNP, apreviously defined recombinant vector, a previously defined host cell,and/or a previously defined antibody.

The invention also relates to the use of a polynucleotide according tothe invention containing at least one previously defined SNP, apreviously defined recombinant vector, a previously defined host cell,and/or a previously defined antibody for the manufacture of atherapeutic compound intended for the prevention or the treatment ofdifferent human disorders and/or diseases such as cancers and tumors,infectious diseases, venereal diseases, immunologically related diseasesand/or autoimmune diseases and disorders, cardiovascular diseases,metabolic diseases, central nervous system diseases, gastrointestinaldisorders, and disorders connected with chemotherapy treatments.

Said cancers and tumors include carcinomas comprising metastasizingrenal carcinomas, melanomas, lymphomas comprising follicular lymphomasand cutaneous T cell lymphoma, leukemias comprising chronic lymphocyticleukemia and chronic myeloid leukemia, cancers of the liver, neck, headand kidneys, multiple myelomas, carcinoid tumors and tumors that appearfollowing an immune deficiency comprising Kaposi's sarcoma in the caseof AIDS.

Said infectious diseases include viral infections comprising chronichepatitis B and C and HIV/AIDS, infectious pneumonias, and venerealdiseases, such as genital warts.

Said immunologically and auto-immunologically related diseases mayinclude the rejection of tissue or organ grafts, allergies, asthma,psoriasis, rheumatoid arthritis, multiple sclerosis, Crohn's disease andulcerative colitis.

Said cardiovascular diseases may include brain injury and anemiasincluding anemia in patients under dialysis in renal insufficiency, aswell as anemia resulting from chronic infections, inflammatoryprocesses, radiotherapies, and chemotherapies.

Said metabolic diseases may include such non-immune associated diseasesas obesity.

Said diseases of the central nervous system may include Alzheimer'sdisease, Parkinson's disease, schizophrenia and depression.

Said diseases and disorders may also include wound healing andosteoporosis.

The compounds of the invention may preferably be used for thepreparation of a therapeutic compound intended to increase theproduction of autologous blood, notably in patients participating in adiffered autologous blood collection program to avoid the use of bloodfrom an other person.

Preferably, the invention concerns the use of a polynucleotide accordingto the invention containing at least one previously defined SNP, apreviously defined recombinant vector, a previously defined host cell,and/or a previously defined antibody for the manufacture of atherapeutic compound intended:

-   -   to prevent or treat anemia, in particular in patients under        dialysis in renal insufficiency, as well as anemia resulting        from chronic infections, inflammatory processes, radiotherapies,        chemotherapies, and/or    -   to increase the production of autologous blood, notably in        patients participating in a differed autologous blood collection        program to avoid the use of blood from an other person, and/or    -   to prevent brain injury.

The dosage of a polypeptide and of the other compounds of the invention,useful as active agent, depends on the choice of the compound, thetherapeutic indication, the mode of administration, the nature of theformulation, the nature of the subject and the judgment of the doctor.

When it is used as active agent, a polypeptide according to theinvention is generally administered at doses ranging between 1 and 300units/kg of the subject.

The invention also has as an object a pharmaceutical composition thatcontains, as active agent, at least one above-mentioned compound such asa polypeptide according to the invention; a hyperglycosylated analog ofthe polypeptide comprising amino acids 28 through 193 of the amino acidsequence SEQ ID NO. 2 and having SNP G104S; a polynucleotide accordingto the invention containing at least one previously defined SNP, apreviously defined recombinant vector, a previously defined host cell,and/or a previously defined antibody, as well as a pharmaceuticallyacceptable excipient.

In these pharmaceutical compositions, the active agent is advantageouslypresent at physiologically effective doses.

These pharmaceutical compositions can be, for example, solids or liquidsand be present in pharmaceutical forms currently used in human medicinesuch as, for example, simple or coated tablets, gelcaps, granules,caramels, suppositories and preferably injectable preparations andpowders for injectables. These pharmaceutical forms can be preparedaccording to usual methods.

The active agent(s) can be incorporated into excipients usually employedin pharmaceutical compositions such as talc, Arabic gum, lactose,starch, dextrose, glycerol, ethanol, magnesium stearate, cocoa butter,aqueous or non-aqueous vehicles, fatty substances of animal or vegetableorigin, paraffinic derivatives, glycols, various wetting agents,dispersants or emulsifiers, preservatives.

The active agent(s) according to the invention can be employed alone orin combination with other compounds such as therapeutic compounds suchas other cytokines such as interleukine or interferons, for example.

The different formulations of the pharmaceutical compositions areadapted according to the mode of administration.

The pharmaceutical compositions can be administered by different routesof administration known to a person skilled in the art.

The invention equally has for an object a diagnostic composition thatcontains, as active agent, at least one above-mentioned compound such asa polypeptide according to the invention, all or part of apolynucleotide according to the invention, a previously definedrecombinant vector, a previously defined host cell, and/or a previouslydefined antibody, as well as a suitable pharmaceutically acceptableexcipient.

This diagnostic composition may contain, for example, an appropriateexcipient like those generally used in the diagnostic composition suchas buffers and preservatives.

The present invention equally has as an object the use:

-   -   a) of a therapeutically effective quantity of a polypeptide        according to the invention, and/or    -   b) of a polynucleotide according to the invention, and/or    -   c) of a host cell from the subject to be treated, previously        defined,        to prepare a therapeutic compound intended to increase the        expression or the activity, in a subject, of a polypeptide        according to the invention.

Thus, to treat a subject who needs an increase in the expression or inthe activity of a polypeptide of the invention, several methods arepossible.

It is possible to administer to the subject a therapeutically effectivequantity of a polypeptide of the invention; of a hyperglycosylatedanalog of the polypeptide comprising amino acids 28 through 193 of theamino acid sequence SEQ ID NO. 2 and having SNP G104S; and/or of theactivating agent and/or activated agent such as previously defined,possibly in combination, with a pharmaceutically acceptable excipient.

It is likewise possible to increase the endogenous production of apolypeptide of the invention by administering a polynucleotide accordingto the invention to the subject. For example, this polynucleotide can beinserted in a retroviral expression vector. Such a vector can beisolated from cells having been infected by a retroviral plasmid vectorcontaining RNA encoding for the polypeptide of the invention, in such afashion that the transduced cells produce infectious viral particlescontaining the gene of interest. See Gene Therapy and other MolecularGenetic-based Therapeutic Approaches, Chapter 20, in Human MolecularGenetics, Strachan and Read, BIOS Scientifics Publishers Ltd (1996).

In accordance with the invention, a polynucleotide containing at leastone coding SNP such as previously defined is going to be preferablyused.

It is equally possible to administer to the subject host cells belongingto him (autologous cells), these host cells having been preliminarilytaken and modified so as to express the polypeptide of the invention, aspreviously described.

The present invention equally relates to the use:

-   -   a) of a therapeutically effective quantity of a previously        defined immunospecific antibody, and/or    -   b) of a polynucleotide permitting inhibition of the expression        of a polynucleotide according to the invention, and/or    -   c) of a host cell from the subject to be treated, as previously        defined        in order to prepare a therapeutic compound intended to reduce        the expression or the activity, in a subject, of a polypeptide        according to the invention.

Thus, it is possible to administer to the subject a therapeuticallyeffective quantity of an inhibiting agent and/or of an antibody such aspreviously defined, possibly in combination, with a pharmaceuticallyacceptable excipient.

It is equally possible to reduce the endogenous production of apolypeptide of the invention by administration to the subject of acomplementary polynucleotide according to the invention permittinginhibition of the expression of a polynucleotide of the invention.

Preferably, a complementary polynucleotide containing at least onecoding SNP such as previously defined can be used.

The present invention concerns also the use of a erythropoietin proteinand/or hyperglycosylated analog for the preparation of a therapeuticcompound for the prevention or the treatment of a patient having adisorder or a disease caused by a EPO variant linked to the presence inthe genome of said patient of a nucleotide sequence having at least 95%identity (preferably, 97% identity, more preferably 99% identity andparticularly 100% identity) with the nucleotide sequence SEQ ID NO. 1,provided that said nucleotide sequence comprises one of the followingSNPs: 465-486 (deletion), c577t, g602c, c1288t, c1347t, t1607c, g1644a,g2228a, g2357a, c2502t, c2621g, g2634a.

Preferably, said therapeutic compound is used for the prevention or thetreatment of one of the diseases selected from the group consisting ofcancers and tumors, infectious diseases, venereal diseases,immunologically related diseases and/or autoimmune diseases anddisorders, cardiovascular diseases, metabolic diseases, central nervoussystem diseases, gastrointestinal disorders, and disorders connectedwith chemotherapy treatments.

Said cancers and tumors include carcinomas comprising metastasizingrenal carcinomas, melanomas, lymphomas comprising follicular lymphomasand cutaneous T cell lymphoma, leukemias comprising chronic lymphocyticleukemia and chronic myeloid leukemia, cancers of the liver, neck, headand kidneys, multiple myelomas, carcinoid tumors and tumors that appearfollowing an immune deficiency comprising Kaposi's sarcoma in the caseof AIDS.

Said infectious diseases include viral infections comprising chronichepatitis B and C and HIV/AIDS, infectious pneumonias, and venerealdiseases, such as genital warts.

Said immunologically and auto-immunologically related diseases mayinclude the rejection of tissue or organ grafts, allergies, asthma,psoriasis, rheumatoid arthritis, multiple sclerosis, Crohn's disease andulcerative colitis.

Said cardiovascular diseases may include brain injury and anemiasincluding anemia in patients under dialysis in renal insufficiency, aswell as anemia resulting from chronic infections, inflammatoryprocesses, radiotherapies, and chemotherapies.

Said metabolic diseases may include such non-immune associated diseasesas obesity.

Said diseases of the central nervous system may include Alzheimer'sdisease, Parkinson's disease, schizophrenia and depression.

Said diseases and disorders may also include wound healing andosteoporosis.

The compounds of the invention may preferably be used for thepreparation of a therapeutic compound intended to increase theproduction of autologous blood, notably in patients participating in adiffered autologous blood collection program to avoid the use of bloodfrom an other person.

Mimetic Compounds of an EPO Polypeptide Comprising the SNP G104S

The present invention also concerns a new compound having a biologicalactivity substantially similar or higher in comparison to that of thepolypeptide of:

-   -   a) amino acid sequence SEQ ID NO. 2, or    -   b) amino acid sequence comprising the amino acids included        between positions 28 and 193 of the amino acid sequence SEQ ID        NO. 2;        provided that said amino acid sequences under a) and b) comprise        the G104S SNP.

Said biological activity may be evaluated, for example, by measuringcellular proliferative activity on cells from murine 32D cell lineover-expressing the EPO receptor, erythroid colony formation or bindingcapacity to EPO receptor.

As mentioned in the experimental part, the G104S mutated EPO increasescellular proliferation of murine 32D cell line over-expressing the EPOreceptor 2 to 5 times more than the wild-type EPO.

As mentioned in the experimental section, the G104S mutated EPO has ahigher capacity to stimulate erythroid colony formation than thewild-type EPO.

As mentioned in the experimental part, the binding capacity of G104Smutated EPO to EPO receptor is higher than that measured with thenatural wild-type EPO.

A new compound of the invention, such as previously defined, may possessa biological activity substantially similar to that of the G104S mutatedEPO, i.e. which is higher than that of the natural wild-type EPO.

Said compound may also have a biological activity which is even higherthan that of the G104S mutated EPO.

A compound according to the invention may have at least one functionassociated with EPO acting upon an EPO receptor and an activitysubstantially similar to that of polypeptide of amino acid sequence SEQID NO. 2 comprising the G104S SNP.

Said compound may also have at least one function associated with EPOacting upon an EPO receptor based on activity induced by effecting achange at said EPO receptor substantially similar to an effect upon suchEPO receptor induced by a polypeptide of amino acid sequence SEQ ID NO.2 comprising the G104S SNP.

Said compound may be a biochemical compound, such as a polypeptide or apeptide for example, or an organic chemical compound, such as asynthetic peptide-mimetic for example.

The present invention also provides a new compound having a cellularproliferative activity on cells from murine 32D cell lineover-expressing the EPO receptor that is 2 to 5 times higher than thatof wild-type EPO.

The present invention also provides a new compound having a highercapacity to stimulate erythroid colony formation than wild-type EPO.

The present invention also provides a new compound having a bindingcapacity to EPO receptor that is higher than that of wild-type EPO.

The present invention also concerns the use of a polypeptide of theinvention containing the G104S SNP, for the identification of a compoundsuch as defined above.

The present invention also concerns a process for the identification ofa compound of the invention, comprising the following steps:

-   -   a) Determining the biological activity, such as stimulating        effect on cell proliferation of 32D cell lines over-expressing        the human EPO-receptor, on erythroid colony formation, and/or        binding capacity to EPO receptor, for example;    -   b) Comparing:        -   i) the activity determined in step a) of the compound to be            tested, with        -   ii) the activity of the polypeptide of amino acid sequence            SEQ ID NO. 2, or of amino acid sequence comprising the amino            acids included between 28 and 193 of the amino acid sequence            SEQ ID NO. 2;            provided that said amino acid sequences comprise the G104S            SNP; and    -   c) Determining, on the basis of the comparison carried out in        step b), whether the compound to be tested has a substantially        similar or higher activity compared to that of the polypeptide        of amino acid sequence SEQ ID NO. 2, or of amino acid sequence        comprising the amino acids included between positions 28 and 193        of the amino acid sequence SEQ ID NO. 2; provided that said        amino acid sequences comprise the G104S SNP.

Preferably, the compound to be tested may be previously identified fromsynthetic peptide combinatorial libraries, high-throughput screening, ordesigned by computer-aided drug design so as to have the samethree-dimensional structure and/or chemical effect as that of thepolypeptide of amino acid sequence SEQ ID NO. 2, or of amino acidsequence comprising the amino acids included between position 28 and 193of the amino acid sequence SEQ ID NO. 2, provided that said amino acidsequences comprise the G104S SNP.

The methods to identify and design compounds are well known by a personskilled in the art.

Publications referring to these methods may be, for example:

-   -   Silverman R. B. (1992). “Organic Chemistry of Drug Design and        Drug Action”. Academic Press, 1st edition (Jan. 15, 1992).    -   Anderson S and Chiplin J. (2002). “Structural genomics; shaping        the future of drug design? Drug Discov. Today. 7(2):105-107.    -   Selick H E, Beresford A P, Tarbit M H. (2002). “The emerging        importance of predictive ADME simulation in drug discovery”.        Drug Discov. Today. 7(2):109-116.    -   Burbidge R, Trotter M, Buxton B, Holden S. (2001). “Drug design        by machine learning: support vector machines for pharmaceutical        data analysis”. Comput. Chem. 26(1): 5-14.    -   Kauvar L. M. (1996). “Peptide mimetic drugs: a comment on        progress and prospects” 14(6): 709.

The compounds of the invention may be used for the preparation of atherapeutic compound intended for the prevention or the treatment of oneof the diseases selected from the group consisting of cancers andtumors, infectious diseases, venereal diseases, immunologically relateddiseases and/or autoimmune diseases and disorders, cardiovasculardiseases, metabolic diseases, central nervous system diseases,gastrointestinal disorders, and disorders connected with chemotherapytreatments.

Said cancers and tumors include carcinomas comprising metastasizingrenal carcinomas, melanomas, lymphomas comprising follicular lymphomasand cutaneous T cell lymphoma, leukemias comprising chronic lymphocyticleukemia and chronic myeloid leukemia, cancers of the liver, neck, headand kidneys, multiple myelomas, carcinoid tumors and tumors that appearfollowing an immune deficiency comprising Kaposi's sarcoma in the caseof AIDS.

Said infectious diseases include viral infections comprising chronichepatitis B and C and HIV/AIDS, infectious pneumonias, and venerealdiseases, such as genital warts.

Said immunologically and auto-immunologically related diseases mayinclude the rejection of tissue or organ grafts, allergies, asthma,psoriasis, rheumatoid arthritis, multiple sclerosis, Crohn's disease andulcerative colitis.

Said cardiovascular diseases may include brain injury and anemiasincluding anemia in patients under dialysis in renal insufficiency, aswell as anemia resulting from chronic infections, inflammatoryprocesses, radiotherapies, and chemotherapies.

Said metabolic diseases may include such non-immune associated diseasesas obesity.

Said diseases of the central nervous system may include Alzheimer'sdisease, Parkinson's disease, schizophrenia and depression.

Said diseases and disorders may also include wound healing andosteoporosis.

The compounds of the invention may preferably be used for thepreparation of a therapeutic compound intended to increase theproduction of autologous blood, notably in patients participating in adiffered autologous blood collection program to avoid the use of bloodfrom an other person.

EXPERIMENTAL SECTION EXAMPLE 1 Modeling of the Protein Encoded by aPolynucleotide of Nucleotide Sequence Containing the SNP g1644a, g2357a,or c2621g and of the Protein Encoded by the Nucleotide Sequence of theWild-type Reference Gene

In a first step, the three-dimensional structure of erythropoietin wasconstructed starting from that available in the PDB database (code IEER)and by using the software Modeler (MSI, San Diego, Calif.). The maturepolypeptide fragment was then modified in such a fashion as to reproducethe mutation D70N, G104S or S147C. A thousand molecular minimizationsteps were conducted on this mutated fragment by using the programsAMBER and DISCOVER (MSI: Molecular Simulations Inc.). Two moleculardynamic calculation runs were then carried out with the same program andthe same force fields. In each case, 50,000 steps were calculated at300° K, terminated by 300 equilibration steps. The result of thismodeling is shown in FIGS. 1, 2, and 3.

EXAMPLE 2 Genotyping of the SNPs g1644a and c262g in a Population ofIndividuals

The genotyping of SNPs is based on the principle of the minisequencingwherein the product is detected by a reading of polarized fluorescence.The technique consists of a fluorescent minisequencing (FP-TDITechnology or Fluorescence Polarization Template-direct Dye-terminatorIncorporation). The minisequencing is performed on a product amplifiedby PCR from genomic DNA of each individual of the population. This PCRproduct is chosen in such a manner that it covers the genic regioncontaining the SNP to be genotyped. After elimination of the PCR primersand the dNTPs that have not been incorporated, the minisequencing iscarried out. The minisequencing consists of lengthening anoligonucleotide primer, placed just upstream of the site of the SNP, byusing a polymerase enzyme and fluorolabeled dideoxynucleotides. Theproduct resulting from this lengthening process is directly analyzed bya reading of polarized fluorescence. All these steps, as well as thereading, are carried out in the same PCR plate.

Thus, the genotyping requires 5 steps:

-   -   1) Amplification by PCR    -   2) Purification of the PCR product by enzymatic digestion    -   3) Elongation of the oligonucleotide primer    -   4) Reading    -   5) Interpretation of the reading        Step 1) Amplification by PCR.

The PCR amplification of the nucleotide sequence of the EPO gene iscarried out starting from genomic DNA coming from 268 individuals ofethnically diverse origins. These genomic DNAs were provided by theCoriell Institute in the United States. The 268 individuals aredistributed as follows: Phylogenic Population Specific Ethnic PopulationTotal % African American African American 50 100.0 Subtotal 50 18.7Amerind South American Andes 10 66.7 South West American Indians 5 33.3Subtotal 15 5.6 Caribbean Caribbean 10 100.0 Subtotal 10 3.7 EuropeanNorth American Caucasian 79 79.8 Caucasoid Iberian 10 10.1 Italian 1010.1 Subtotal 99 36.9 Mexican Mexican 10 100.0 Subtotal 10 3.7 NortheastAsian Chinese 10 50.0 Japanese 10 50.0 Subtotal 20 7.5 Non-EuropeanGreek 8 21.6 Caucasoid Indo-Pakistani 9 24.3 Middle-Eastern 20 54.1Subtotal 37 13.8 Southeast Asian Pacific Islander 7 41.2 South Asian 1058.8 Subtotal 17 6.3 South American South American 10 100.0 Subtotal 103.7 Total 268 100*Phylogenic populations are adapted from: Cavalli-Sforza, P. Menozzi,and A. Piazza. 1994. “The History and Geography of Human Genes.”Princeton: Princeton University Press. pp 80.

The genomic DNA coming from each one of these individuals constitutes asample.

The PCR amplification is carried out from primers which can easily bedesigned by the person skilled in the art on the basis of the nucleotidesequence SEQ ID NO. 1.

For the genotyping of g1644a, the PCR amplification is carried out usingthe following primers: SEQ ID NO.5: Sense primer: TTCAGGGACCCTTGACTC SEQID NO.6: Antisense primer: GATCATTCTCCCTTTCATC CThese nucleotide sequences permit amplification of a fragment of alength of 208 nucleotides, from the nucleotide 1557 to the nucleotide1764 in the nucleotide sequence SEQ ID NO. 1.

For the genotyping of c2621g, the PCR amplification is carried out usingthe following primers: SEQ ID NO.7: Sense primer: TTGCATACCTTCTGTTTGC TSEQ ID NO.8: Antisense primer: CACAAGCAATGTTGGTGAGThese nucleotide sequences permit amplification of a fragment of alength of 626 nucleotides, from the nucleotide 2192 to the nucleotide2817 in the nucleotide sequence SEQ ID NO. 1.

For each SNP to be genotyped, the PCR product will serve as a templatefor the minisequencing.

The total reaction volume of the PCR reaction is 5 μl per sample. Thisreaction volume is composed of the reagents indicated in the followingtable: Initial Vol. per Final Supplier Reference Reactant Conc. tube(μl) Conc. Life Technology Delivered w/Taq Buffer (X) 10 0.5 1 LifeTechnology Delivered w/Taq MgSO₄ (mM) 50 0.2 2 AP Biotech 27-2035-03dNTPs (mM) 10 0.1 0.2 On request Sense Primer (μM) 10 0.1 0.2 On requestAntisense Primer (μM) 10 0.1 0.2 Life Technology 11304-029 Taq platinum5 U/μl 0.02 0.1 U/rxn H₂O Qsp 5 μl 1.98 DNA (sample) 2.5 ng/μl 2 5ng/rxn Total volume 5 μlThese reagents are distributed in a black PCR plate having 384 wellsprovided by ABGene (ref:TF-03 84-k). The plate is sealed, centrifuged,then placed in a thermocycler for 384-well plates (Tetrad of MJResearch) and undergoes the following incubation: PCR Cycles: 1 min at94° C., followed by 36 cycles composed of 3 steps (15 sec. at 94° C., 30sec. at 56° C. 1 min at 68° C.).Step 2) Purification of the PCR Product by Enzymatic Digestion.

The PCR amplified product is then purified using two enzymes: ShrimpAlkaline Phosphatase (SAP) and exonuclease I (Exo I). The first enzymepermits the dephosphorylation of the dNTPs which have not beenincorporated during the PCR amplification, whereas the second eliminatesthe remaining single stranded DNA, in particular the primers which havenot been used during the PCR. This digestion is done by addition, ineach well of the PCR plate, of a reaction mixture of 5 μl per sample.

This reaction mixture is composed of the following reagents: InitialVol./tube Supplier Reference Reactant Conc. (μl) Final conc. AP BiotechE70092X SAP 1 U/μl 0.5 0.5/rxn AP Biotech 070073Z Exo I 10 U/μl 0.1  1/rxn AP Biotech Supplied w/SAP Buffer SAP (X) 10 0.5 1 H₂O Qsp 5 μl3.9 PCR product 5 μl Total vol. 10 μlOnce filled, the plate is sealed, centrifuged, then placed in athermocycler for 384-well plates (Tetrad of MJ Research) and undergoesthe following incubation: Digestion SAP-EXO: 45 min at 37° C., 15 min at80° C.Step 3) Elongation of the Oligonucleotide Primer

The elongation or minisequencing step is then carried out on thisdigested PCR product by addition of a reaction mixture of 5 pl perprepared sample, as indicated in the following table: Initial Vol. perFinal Supplier Reference Reactant conc. tube (μl) conc. Own ElongationBuffer¹  5 1 1 preparation (X) Life On request Miniseq Primer (μM) 100.5 1 Technologies A or B AP Biotech 27-2051 ddNTPs² (μM) 2.5 0.25 0.125(61, 71, 81)-01 2 are non labeled of each of each NEN Nel 472/5 ddNTPs²(μM) 2.5 0.25 0.125 and Nel 492/5 2 are labeled with of each of eachTamra and R110 AP Biotech E79000Z Thermo-sequenase 3.2 U/μl 0.125 0.4 U/reaction H₂O Qsp 5 μl 3.125 digested PCR product 10 Total volume 15¹The 5X elongation buffer is composed of 250 mM Tris-HCl pH 9, 250 mMKCl, 25 mM NaCl, 10 mM MgCl₂ and 40% glycerol.²For the ddNTPs, a mixture of the 4 bases is carried out according tothe polymorphism studied. Only the 2 bases of interest (C/T for g1644aread in antisense or C/G for c2621g) composing the functional SNP arelabeled, either in Tamra, or in R110.In the case of the genotyping of g1644a, the mixture of ddNTPs iscomposed of:2.5 μM of ddGTP non labeled,2.5 μM of ddATP non-labeled,2.5 μM of ddTTP (1.875 μM of ddTTP non labeled and 0.625 μM of ddTTPTamra labeled),2.5 μM of ddCTP (1.875 μM of ddCTP non labeled and 0.625 μM of ddCTPR110 labeled).In the case of the genotyping c2621g, the mixture of ddNTPs is composedof:2.5 μM of ddATP non labeled,2.5 μM of ddTTP non-labeled,2.5 μM of ddGTP (1.875 μM of ddGTP non labeled and 0.625 μM of ddGTPTamra labeled),2.5 μM of ddCTP (1.875 μM of ddCTP non labeled and 0.625 μM of ddCTPR110 labeled).

The sequences of the two minisequencing primers necessary for thegenotyping were determined in a way to correspond to the sequence of thenucleotides located upstream of the site of a SNP according to theinvention. The PCR product that contains the SNP being a double strandedDNA product, the genotyping can therefore be done either on the sensestrand or on the antisense strand. The selected primers are manufacturedby Life Technologies Inc.

For the SNP g1644a, the minisequencing primers tested are the following:

-   SEQ ID NO. 9: Sense primer (A): tgcagcttgaatgagaatatcactgtccca-   SEQ ID NO. 10: Antisense primer (B):    cctcttccaggcatagaaattaactttggtgt

The minisequencing of the SNP g1644a was first validated over 48samples, then genotyped over the set of the population of individualscomposed of 268 individuals and 11 negative controls. Severalminisequencing conditions were tested and the following optimalcondition was retained for the genotyping of g1644a:

-   Antisense primer+ddCTP-R110+ddTTP-Tamra

For the SNP c2621g, the minisequencing primers tested are the following:

-   SEQ ID NO. 11: Sense primer (A): ttggcagaaggaagccatct-   SEQ ID NO. 12: Antisense primer (B): ctgaggccgcatctggaggg

The minisequencing of the SNP c2621g was first validated over 48samples, then genotyped over the set of the population of individualscomposed of 268 individuals and 10 negative controls. Severalminisequencing conditions were tested and the following optimalcondition was retained for the genotyping of c2621g:

-   Sense primer+ddCTP-R110+ddGTP-Tamra    Once filled, the plate is sealed, centrifuged, then placed in a    thermocycler for 384-well plates (Tetrad of MJ Research) and    undergoes the following incubation: Elongation cycles: 1 min. at 93°    C., followed by 35 cycles composed of 2 steps (10 sec. at 93° C., 30    sec. at 55° C.).

After the last step in the thermocycler, the plate is directly placed ona polarized fluorescence reader of type Analyst® HT of LJL BiosystemsInc. The plate is read using Criterion Host® software by using twomethods. The first permits reading the Tamra labeled base by usingemission and excitation filters specific for this fluorophore(excitation 550-10 nm, emission 580-10 nm) and the second permitsreading the R110 labeled base by using the excitation and emissionfilters specific for this fluorophore (excitation 490-10 nm, emission520-10 nm). In the two cases, a dichroic double mirror (RI 10/Tamra) isused and the other reading parameters are:

-   -   Z-height: 1.5 mm    -   Attenuator: out    -   Integration time: 100,000 μsec.    -   Raw data units: counts/sec    -   Switch polarization: by well    -   Plate settling time: 0 msec    -   PMT setup: Smart Read (+), sensitivity 2    -   Dynamic polarizer: emission    -   Static polarizer: S

A file result is thus obtained containing the calculated values of mP(milliPolarization) for the Tamra filter and that for the R110 filter.These mP values are calculated from the intensity values obtained on theparallel plane (//) and on the perpendicular plane (⊥) according to thefollowing formula:mP=1000(//−g⊥)/(//+g⊥).

In this calculation, the value ⊥ is weighted by a factor g. It is amachine parameter that must be determined experimentally beforehand.

Steps 4) and 5) Interpretation of the Reading and Determination of theGenotypes.

The mP values are reported on a graph using Microsoft Inc. Excelsoftware, and/or Allele Caller(® software developed by LJL BiosystemsInc.

On the abscissa is indicated the mP value of the Tamra labeled base, onthe ordinate is indicated the mP value of the R110 labeled base. Astrong mP value indicates that the base labeled with this fluorophore isincorporated and, conversely, a weak mP value reveals the absence ofincorporation of this base.

Up to three homogenous groups of nucleotide sequences having differentgenotypes are obtained.

The use of the Allele Caller® software permits, once the identificationof the different groups is carried out, to directly extract the genotypedefined for each individual in table form.

Results of the Minisequencing for the SNPs R1644a and c2621 g.

After the completion of the genotyping process, the determination of thegenotypes of the individuals of the population of individuals for thetwo functional SNPs studied here was carried out using the graphsdescribed above.

For the SNP g1644a, this genotype is in theory either homozygote GG, orheterozygote GA or homozygote AA in the tested individuals. In reality,and as shown below, the homozygote genotype AA is not detected in thepopulation of individuals.

Similarly, for the SNP c2621g, this genotype is in theory eitherhomozygote CC, or heterozygote CG, or homozygote GG in the testedindividuals. In reality, and as shown below, the homozygote genotype GGis not detected in the population of individuals.

The results of the negative controls, of the distribution of thedetermined genotypes in the population of individuals and thecalculation of the different allelic frequencies for these twofunctional SNPs are presented in the following tables: Number of Numberof individuals controls Percentage tested genotyped tested genotyped ofsuccess g1644a 268 267 11 11 99.6 c2621g 268 250 10 10 93.5

g1644a (D70N) Phylogenic Population Total f (95% CI) GG % GA % AA %Total African American 50 50 100 50 Amerind 15 15 100 15 Caribbean 10 10100 10 European Caucasoid 99 0.5 (0, 1.5) 97 99.0 1 1.0 98 Mexican 10 10100 10 Non-European Caucasoid 37 37 100 37 Northeast Asian 20 20 100 20South American 10 10 100 10 Southeast Asian 17 17 100 17 Total 268 0.2(0, 0.6) 266 99.6 1 0.4 267 c2621g (S147C) Phylogenic Population Total f(95% CI) CC % CG % GG % Total African American 50 50 100 50 Amerind 1515 100 15 Caribbean 10 10 100 10 European Caucasoid 99 0.5 (0, 1.6) 9198.9 1 1.1 92 Mexican 10 8 100 8 Non-European Caucasoid 37 32 100 32Northeast Asian 20 19 100 19 South American 10 8 100 8 Southeast Asian17 16 100 16 Total 268 0.2 (0, 0.6) 249 99.6 1 0.4 250In the above table.N represents the number of individuals.% represents the percentage of individuals in the specificsub-population.the allelic frequency represents the percentage of the mutated allele inthe specific sub-population.95% IC represents the minimal and maximal interval of confidence at 95%.

By examining these results by population, it is observed that, in thecase of SNP g1644a, the only heterozygote individual GA comes from thesub-population European Caucasoid of the population of individuals.

Similarly, by examining these results by population, it is observedthat, in the case of SNP c2621g, the only heterozygote individual CGcomes from the sub-population European Caucasoid of the population ofindividuals.

EXAMPLE 3 Study of the Biological Function of G104S MutatedErythropoietin Compared to that of Natural Wild-type Erythropoietin

The first step consists of preparing mutated and wild-type EPO proteins

a) Cloning of the Natural Wild-type Erythropoietin and MutatedErythropoietin (g2357a) in the Eukaryotic Expression VectorpcDNA3.1/His-Topo Carrying the Geneticin-resistance Gene

In comparison to the sequence of the erythropoeitin protein published inSwissProt, the polyhistidine tagged EPO cDNA from the Genestorm clone(H-X02158M—Invitrogen) harbored the K143E (G₄₂₇AG) mutation (the numberin subscript corresponds to the nucleotide position on the cDNAsequence). Thus, we first restituted the natural wild-type E143 (A₄₂₇AG)sequence using the Exsite PCR kit (Stratagene) and the followingprimers:

-   SEQ ID NO. 13: Sense primer: CCAGAAGGAAGCCATCTCCCCT-   SEQ ID NO. 14: Antisense primer (phosphorylated on the 5′ end):    GCTCCCAGAGCCCGAAGCAG

In parallel, the G104S (G₃₁₀GC=>AGC) mutated erythropoietin was obtainedusing the Exsite PCR kit (Stratagene corp.) and the following primers:

-   SEQ ID NO. 15: Sense primer: CGGAGCCAAGCCCTGTTGGTCA-   SEQ ID NO. 16: Antisense primer (phosphorylated on the 5′ end):    CAGGACAGCTTCCGACAGCA

To remove the polyhistidine tail and isolate the nucleotide sequencescorresponding to the complete EPO protein (i.e. natural signal peptideand mature protein), whether mutated or wild-type form, a PCRamplification was carried out using the following primers: SEQ ID NO.17:Sense primer: ATGGGGGTGCACGAATGT TCC SEQ ID NO.18: Antisense primer:TCATCTGTCCGCTGTCCT GC

The PCR products are inserted in the eukaryotic expression vectorpcDNA3.1/GS/HisTopo (TOPO™-cloning; Invitrogen Corp.) under the controlof the CMV promoter. This vector allows the constitutive expression ofproteins in eukaryotic cell lines.

After checking of the nucleotide sequence of the vector region codingfor the recombinant proteins, the different recombinant expressionvectors are transfected into the Chinese Hamster Ovary cells (CHO) usingSuperfect (QIAgen).

b) Selection of Clones Over-expressing Natural Wild-type or Mutated EPO

Two days after the transfection with the various EPO constructs, the CHOcells are placed in a culture medium containing 800 μg/ml of Geneticin(Invitrogen). As a result of a 2-week growth in these cultureconditions, stable cells over-expressing EPO are selected. The cells arethen cloned by the limited dilution method. Thirty clones from cellstransfected with either wild-type or mutated EPO are screened forexpression of the EPO protein using an EPO ELISA (R&D Systems). SeveralEPO-expressing clones are picked and kept frozen. Among them, the cloneproducing the highest amount of either wild-type or mutated EPO was usedfor EPO mass production.

c) Purification of EPO Proteins

After EPO expression in the CHO culture, the culture medium iscentrifuged at 1500 rpm for 20 minutes permitting recovery of thesupernatant. The supernatant is then concentrated 10 times usingLabscale (Millipore membrane 5 Kda), dialyzed against 3 liters of bufferTris 50 mM, NaCl 25 mM pH 9 and purified on an anion exchange column(Pharmacia, HiprepQ). After protein elution using a step at 200 mM NaCl,the protein is desalted against buffer NaH₂PO₄ 50 mM, NaCl 25 mM, pH 7and purified on Heparine HP (Pharmacia). Protein elution is then carriedout using a step at 150 mM NaCl. Finally, the EPO protein is analyzed bySDS-PAGE gel characterization followed by a quantification usingdensitometry (Biorad densitometer GS800).

The second step consists of preparing 32D murine cells over-expressingthe EPO receptor.

d) Cloning of the Natural EPO Receptor in the Eukaryotic ExpressionVector pcDNA3.1/GS/HisTopo Carrying the Zeocyn-resistance Gene:

To further insert the cDNA in frame with the V5 epitope and apolyhistidine tail, the complete sequence of the natural human EPOreceptor cDNA from the Genestorm clone (H-M60459M—Invitrogen) isamplified by PCR using the following primers: SEQ ID NO.19: Senseprimer: ATGGACCACCTCGGGGCG TC SEQ ID NO.20: Antisense primer:AGAGCAAGCCACATAGCT GGGGG

The PCR product is inserted into the eukaryotic expression vectorpcDNA3.1/GS/HisTopo (TOPO™-cloning; Invitrogen Corp.) under the controlof the CMV promoter. This vector allows constitutive expression ofproteins in eukaryotic cells lines. In this case, the EPO receptor istagged with an additional C-terminal sequence containing apoly-histidine tail and a V5 epitope. After checking of the nucleotidesequence of the vector region coding for the recombinant receptor, theconstruct was electroporated into the murine 32D cell line (ATCC)

e) Selection of Stable Cells Over-expressing the EPO-Receptor.

To select stable cells over-expressing the human EPO-Receptor, the 32Dcell line electroporated with the construct encoding the EPO-Receptorwas cultivated in the presence of 200 μg/ml of Zeocin (Invitrogen) for 5weeks before its ability to proliferate in the presence of commercialhuman EPO (R&D Systems) was assessed.

Finally, the biological effect of mutated EPO and wild-type EPO isdetermined by two different tests: by evaluation of the ability of thedifferent EPO proteins to induce cell proliferation of murine 32 cellsover-expressing the EPO receptor and by measurement of the directbinding of mutated EPO and wild-type EPO to EPO receptor.

f) Evaluation of the Ability of Wild-type and Mutated G104S EPO toInduce Cell Proliferation of Murine 32D Cells Over-expressing theEPO-Receptor.

The ability of wild-type EPO and G104S mutated EPO to induce cellproliferation is assessed on murine 32D cells over-expressing theEPO-Receptor (32D-EPOR cells). This test was performed first on proteinextracts containing the different EPO proteins produced in the previoussteps, and, second, on purified EPO proteins obtained as previouslydescribed.

The principle is that 32D-EPOR cells are inoculated in a 96-well plateat a cell density of 2.10⁴ cells/well in a 200 μl final culture mediumcontaining 10% fetal calf serum. 32D-EPOR cells are incubated withserial dilutions of either wild-type or mutated EPO (from 0.024 to 140ng/ml in the case of protein extracts and from 0.76×10⁻³ to 400 ng/ml inthe case of purified EPO), at 37° C., for 5 days after which Uptiblue(Uptima) is added to the cultures. The rate of cell proliferation isquantified by measuring the fluorescence emitted at 590 nm (excitation560 nm) after an additional period of incubation of 24 hours in the caseof protein extracts and 4 hours in the case of purified EPO.

The proliferative activity of the natural wild-type and the mutated EPOis based on the determination of the EC50 value corresponding to the EPOconcentration (ng/ml) for which cell proliferation reaches 50%.

First, two experiments such as described above have been carried outusing the proteins extracts containing EPO, each experiment beingrepeated three times. The results of these experiments are representedin FIG. 4A and FIG. 4B, respectively. In FIG. 4, for each proteinconcentration, the points correspond to the average of the threemeasures and the standard deviation represents the variation between thethree repeats.

The EC values obtained from these curves are the following:

-   -   in the first experiment: 24.22 ng/ml for the wild-type EPO and        4.68 ng/ml for the G104S mutated EPO    -   in the second experiment: 5.24 ng/ml for the wild-type EPO and        3.7 ng/ml for the G104S mutated EPO

Thus, FIGS. 4A and 4B and the EC50 values indicate that the G104Smutated EPO stimulating effect on cell proliferation of 32D cell linesover-expressing the human EPO-Receptor is 2 to 5 times higher than thatof the natural wild-type EPO.

Second, similar experiments have been carried out using purified EPOproteins. The results of two experiments, performed in triplicates, arerepresented in FIG. 5A and FIG. 5B, respectively. In FIG. 5, for eachprotein concentration, the points correspond to the average of the threemeasures and the standard deviation represents the variation between thethree repeats.

The EC50 values obtained from these curves are the following:

-   -   in the first experiment: 2.38 ng/ml for the wild-type EPO and        0.58 ng/ml for the G1104S mutated EPO    -   in the second experiment: 2.57 ng/ml for the wild-type EPO and        1.12 ng/ml for the G104S mutated EPO.

Thus, FIGS. 5A and 5B and the EC50 values indicate that the purifiedG104S mutated EPO stimulating effect on cell proliferation of 32D celllines over-expressing the human EPO-Receptor is 2 to 5 times higher thanthat of the purified natural wild-type EPO, confirming the resultsobtained with the protein extracts.

g) Stimulation of Erythroid Colony Formation by G104S MutatedErythropoietin

The capacity of G104S mutated erythropoietin to stimulate erythroidcolony formation was evaluated and compared to that of wild-typeerythropoietin.

To do so, human bone marrow cells from healthy individuals werecollected and separated on a ficoll gradient. Nucleated cells (2.5×10⁵cells) were plated in semisolid methyl cellulose. Mutated or wild-typeerythropoietin ranging from 0.25 to 10 ng/mL was then added to theculture medium. After 10 days of culture, erythroid colonies werecounted.

This experiment was performed twice and the average results arecollected in the following table and represented in FIG. 6. Number ofcolonies EPO (ng/mL) Wild-type EPO G104S EPO 0.25 170 230 0.5 505 685 1540 810 2.5 620 860 5 670 855 10 715 950

These data clearly demonstrate that G104S mutated erythropoietinstimulates erythroid colony formation. In particular, stimulation oferythroid colony formation by G104S mutated erythropoietin is 30 to 50%higher than that measured with wild-type erythropoietin.

h) Interaction Between EPO and the EPO Receptor

The interaction between EPO and its receptor (EPO-R) was determinedusing Surface Plasmon Resonance technology (Biacore, SPR).

To compare the affinities of G104S mutated EPO and wild-type EPO,quantitative measurements of the binding interaction between EPO and theextra-cellular part of the EPO-R are carried out using the EPO-R targetligand immobilized on a sensor chip surface and then passing, on thechip, different concentrations of an analyte consisting of the EPOproteins to be tested.

The carboxymethylated dextran layer of the chip is designed to bindnickel to mediate the capture of ligands via metal chelation of apoly-histidine tail.

For this reason, we designed an EPO-Receptor corresponding to theextra-cellular part of the mature human receptor (amino-acids 25-247)followed by a C-terminal V5 epitope and a poly-histidine tail(KGFSFNWGGKPIPNPLLGLDSTGVDHHHHHH-C-ter). The corresponding cDNA fragmentwas inserted into the Pichia pastoris vector pPICZalpha his-topo(Invitrogen) using the following specific oligonucleotides: SEQ IDNO.21: Sense primer: GCGCCCCCGCCTAACCT C SEQ ID NO.22: Antisense primer:GTCGCTAGGCGTCAGCA GCGA

Two saturated pre-cultures of 50 ml of BMGY medium (2% Peptone, 1% yeastextract, 1.34% YNB, 1% Glycerol, 100 mM potassium phosphate, 0.4mg/Liter biotin pH 6.8) containing a clone coding for natural wild-typeEPO or that coding for G104S EPO, were carried out for 24 hours at 30°C. at an agitation of 200 rotations per minute (rpm).

When the culture reaches a cellular density corresponding to an opticaldensity of 5.0 measured at a wavelength of 600 nm, it is used toinoculate, at 1/5, 200 ml of BMMY medium (2% Peptone, 1% yeast extract,1.34% YNB, 0.5% Methanol, 100 mM potassium phosphate, 0.4 mg/L biotin pH6.8).

The expression of the protein is then induced by methanol at a finalconcentration of 0.5%, for 2 to 5 days at 30 ° C., with an agitation ofthe culture flask at 200 rpm.

The supernatant containing about 10 mg/ml of EPO-R is concentrated byultra-filtration onto a labscale apparatus (cut-off 5000 Da) and bufferis exchanged by dialysis against sodium phosphate 50 mM, Tris(Cl) 10 mM,pH 8,0, NaCl 150 mM, imidazol 10 mM. Poly-histidine EPO-R is thencaptured onto a Hi-Trap pre-loaded with nickel-sulfate (AmershamPharmacia). Fractions containing the protein were desalted using a gelfiltration column (buffer Tris(Cl) pH 9, NaCl 50 mM) and then purifiedat about 95% onto an anionic exchange chromatography. Purity andconcentration were estimated using SDS-PAGE gels.

The sensor chip NTA is then activated passing over nickel sulfate 500 μMwith a flow of 20 μl/min. The EPO-R is then captured onto the surface ata concentration of 50 nM in a HBS-P buffer (10 mM HEPES, NaCl 150 mM,0,005% P20 EDTA 50 μM) with a flow of 10 μ/min. Concentrations ofwild-type EPO and G104S mutated EPO ranging from 0.45 to 15 nM were thenpassed over the sensor chip. A regeneration using HBS-P, EDTA 0,35 M wasperformed after each concentration test. An automatic procedurepermitted to evaluate the binding interaction of the wild-type EPO andG104S mutated EPO for the six concentrations in the range indicatedabove.

FIG. 7 shows the results of the binding measurements for twoconcentrations (7.5 and 15 nM) of G104S mutated EPO and wild-type EPO.

These results indicate that the G104S mutated EPO binds more quickly toits receptor than the wild-type EPO, confirming the effect observed atthe cellular level (see examples described in 3f and 3g ). As aconsequence, this demonstrates that the strong positive effect of G104Smutated EPO on proliferation of murine 32D cells over-expressing the EPOreceptor is related, at least in part, to a better affinity of G104Smutated EPO to its receptor.

This effect on EPO potency of a mutation affecting the amino acid atposition 104 in the immature EPO protein sequence is extremelysurprising. Indeed, the crystal structure of EPO complexed to the EPOreceptor indicates that only the three helices A, C, and D of EPO (outof the four helices A, B, C, and D) are involved in the binding with EPOreceptor (Syed et al. Efficiency of signaling through cytokine receptorsdepends critically on receptor orientation. Nature 395:511-516(1998)).In addition, site-directed mutagenesis analyzing the structure-functionrelationship in EPO demonstrates that changes in amino acids situated inhelix B, in the neighborhood of residue 77, have no substantial effecton EPO activity (Eliott et al. Mapping of the active site of recombinanthuman erythropoietin. Blood. 89: 493-502 (1997); Wen et al.Erythropoietin structure-function relationships. Identification offunctionally important domains. J. Biol. Chem. 269:22839-22846(1994)).

Such novel information on structure/function of EPO could also be usedto identify, design and develop new EPO-like entities (either chemicalor peptidic) that mimic EPO activity on its human receptor.

1. An isolated polynucleotide comprising a nucleotide sequence at least90% identical to SEQ ID NO. 1, wherein said nucleotide further comprisesat least one SNP selected from the group consisting of 465-486(deletion), c577t, g602c, c1288t, c1347t, g1644a, g2228a, g2357a,c2502t, c2621 g, and g2634a.
 2. The isolated polynucleotide of claim 1,wherein said polynucleotide comprises a nucleotide sequence at least 90%identical to nucleotides 615 to 2763 of SEQ ID NO. 1, and wherein saidnucleotide sequence comprises at least one coding SNP selected from thegroup consisting of g1644a, g2357a, and c2621g.
 3. The isolatedpolynucleotide of claim 1, wherein said polynucleotide comprises the SNPg2357a.
 4. An isolated polynucleotide consisting of a part of thepolynucleotide of claim 1, wherein said polynucleotide comprises atleast 10 nucleotides and at least one SNP selected from the groupconsisting of g1644a, g2357a, and c2621g.
 5. The isolated polynucleotideof claim 1, wherein said nucleotide sequence is at least 95% identicalto SEQ ID NO.
 1. 6. The isolated polynucleotide of claim 1, wherein saidnucleotide sequence is at least 99% identical to SEQ ID NO.
 1. 7. Theisolated polynucleotide of claim 2, wherein said nucleotide sequence isat least 95% identical to nucleotides 615 to 2763 of SEQ ID NO.
 1. 8.The isolated polynucleotide of claim 2, wherein said nucleotide sequenceis at least 99% identical to nucleotides 615 to 2763 of SEQ ID NO.
 1. 9.The isolated polynucleotide of claim 2, wherein said polynucleotidecomprises the SNP g2357a.
 10. An isolated polynucleotide consisting of apart of the polynucleotide of claim 2, wherein said polynucleotidecomprises at least 10 nucleotides and at least one SNP selected from thegroup consisting of g1644a, g2357a, and c2621g.
 11. The isolatedpolynucleotide of claim 10, wherein said polynucleotide comprises theSNP g2357a.
 12. An isolated polynucleotide encoding a polypeptide,wherein said polypeptide comprises the amino acid sequence SEQ ID NO. 2or a fragment thereof, said polypeptide further comprising at least onecoding SNP selected from the group consisting of D70N, G104S, and S147C.13. The isolated polynucleotide of claim 12, wherein said polypeptidecomprises the SNP G104S.
 14. An isolated polynucleotide, wherein saidpolynucleotide comprises a nucleotide sequence that is complementary toa polynucleotide according to claim
 2. 15. An isolated polynucleotide,wherein said polynucleotide comprises a nucleotide sequence that iscomplementary to a polynucleotide according to claim
 9. 16. An isolatedpolynucleotide, wherein said polynucleotide comprises a nucleotidesequence that is complementary to a polynucleotide according to claim10.
 17. An isolated polynucleotide, wherein said polynucleotidecomprises a nucleotide sequence that is complementary to apolynucleotide according to claim
 11. 18. A recombinant vectorcomprising a polynucleotide according to claim
 2. 19. A recombinantvector comprising a polynucleotide according to claim
 9. 20. A host cellcomprising a recombinant vector according to claim
 18. 21. A host cellcomprising a recombinant vector according to claim
 19. 22. A method forseparating a polypeptide, comprising cultivating a host cell accordingto claim 19 in a culture medium and separating said polypeptide from theculture medium.
 23. The polypeptide encoded by the isolatedpolynucleotide of claim 2 or claim
 9. 24. A method for preventing ortreating in an individual a disease selected from the group consistingof cancers and tumors, infectious diseases, venereal diseases,immunologically related diseases and autoimmune diseases and disorders,cardiovascular diseases, metabolic diseases, central nervous systemdiseases, gastrointestinal disorders, and disorders connected withchemotherapy treatments, said method comprising administering to saidindividual a therapeutically effective amount of an agent comprising apolynucleotide of any of claims 1 or 2, plus a pharmaceuticallyacceptable excipient.
 25. A method for preventing or treating anemias inan individual, said method comprising administering to said individual atherapeutically effective amount of an agent comprising a polynucleotideof any of claims 1 or 2, plus a pharmaceutically acceptable excipient.26. The method of claim 25, wherein said anemias include anemia inpatients under dialysis in renal insufficiency, as well as anemiaresulting from chronic infections, inflammatory processes,radiotherapies, and chemotherapies.
 27. A method for increasing theproduction of autologous blood, notably in patients participating in adiffered autologous blood collection program, comprising administeringto said individual a therapeutically effective amount of an agentcomprising a polynucleotide of claim 1 or claim 2, plus apharmaceutically acceptable excipient.
 28. A method for preventing ortreating in an individual a disease selected from the group consistingof cancers and tumors, infectious diseases, venereal diseases,immunologically related diseases and autoimmune diseases and disorders,cardiovascular diseases, metabolic diseases, central nervous systemdiseases, gastrointestinal disorders, and disorders connected withchemotherapy treatments, said method comprising administering to saidindividual a therapeutically effective amount of an agent comprising apolynucleotide of claim 9, plus a pharmaceutically acceptable excipient.29. A method for preventing or treating anemias in an individual, saidmethod comprising administering to said individual a therapeuticallyeffective amount of an agent comprising a polynucleotide of claim 9,plus a pharmaceutically acceptable excipient.
 30. The method of claim29, wherein said anemias include anemia in patients under dialysis inrenal insufficiency, as well as anemia resulting from chronicinfections, inflammatory processes, radiotherapies, and chemotherapies.31. A method for increasing the production of autologous blood, notablyin patients participating in a differed autologous blood collectionprogram, comprising administering to said individual a therapeuticallyeffective amount of an agent comprising a polynucleotide of claim 9,plus a pharmaceutically acceptable excipient.